Efficient method for reprogramming blood to induced pluripotent stem cells

ABSTRACT

Described herein are methods and compositions related to generation of induced pluripotent stem cells (iPSCs). Improved techniques for establishing highly efficient, reproducible reprogramming using non-integrating episomal plasmid vectors. Using the described reprogramming protocol, one is able to consistently reprogram non-T cells with close to 100% success from non-T cell or non-B cell sources. Further advantages include use of a defined reprogramming media E7 and using defined clinically compatible substrate recombinant human L-521. Generation of iPSCs from these blood cell sources allows for recapitulation of the entire genomic repertoire, preservation of genomic fidelity and enhanced genomic stability.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/184,241, filed Jun. 16, 2016, which issued as U.S. Pat. No.10,221,395.

FIELD OF INVENTION

Described herein are methods and compositions related to regenerativemedicine including the derivation of induced pluripotent stem cells(iPSCs) from whole blood and peripheral blood, such methods andcompositions that provide a renewable source of transplant material.

BACKGROUND

Pluripotent stem cells (“pSCs”) present broad opportunities to generatetherapeutic materials for use in regenerative medicine, as well asproviding invaluable in vitro models for studying disease initiation andprogression. One category of pSCs, induced pluripotent stem cells(“iPSCs”), possess the hallmark stem cell properties of self-renewal(i.e., immortal) and differentiation capacity into cells derived fromall three embryonic germ layers (i.e., pluripotency). These cells can beobtained through “reprogramming”, which involves dedifferentiation ofcells from non-embryonic sources, such as adult somatic cells. Thereprogramming process obviates potential ethical concerns over embryonicsource material for other types such pSCs, such as embryonic stem cells(“ESCs”), while providing a further benefit of enabling potentialpatient-specific immunological incompatibility.

In addition to establishing robust reprogramming techniques, fullrealization of therapeutic goals for stem-cell regenerative medicinefurther requires consideration of the types of host cells that can serveas a resource for renewable regenerative material. Ideally, cells wouldpossess not only the requisite plasticity for successful reprogrammingand stability in subsequent propagation, but also provide advantages inclinical aspects, such as ease of isolation, storage, stability andmaintenance. In this regard, blood cells represent an attractive choicefor such uses, given their wide use medical diagnostics and as a highlyaccessible resource for cellular reprogramming. Whereas fibroblasts havebeen a widely used cellular source for many reprogramming experimentsperformed in the last decade, this source material but may not be thebest choice for directed reprogramming. Skin biopsy to obtainfibroblasts is an invasive, non-sterile procedure requiring expansion ofharvested cells before experimentation. Most importantly, skin cellsharbor more mutations due to environmental insults such as UVirradiation than cells from inside the body such as blood.

Given the eventual therapeutic goal of generating patient-specific,immunocompatible biological material, there is a great need in the artto establish a robust and reproducible means for reprogramming cells,along with identifying sources of therapeutic materials suitable foreventual clinical application. Such improved methods would need topossess high efficiency of reprogramming, consistent reproduction,produce cells possessing genomic stability and be readily extendible toa variety of cell types.

Described herein are improved techniques for establishing highlyefficient, reproducible reprogramming using non-integrating episomalplasmid vectors, including generation of iPSCs from non-T cell, non-Bcell component in blood samples. These described approaches allow foruse of blood as a readily accessible resource for cellular reprogrammingwith superior properties in genomic and karyotype stability.

SUMMARY OF THE INVENTION

Described herein is a method of generating blood cell derived inducedpluripotent stem cells, comprising providing a quantity of blood cells,delivering a quantity of reprogramming factors into the blood cells, andculturing the blood cells in a reprogramming media for at least 4 days,wherein delivering the reprogramming factors, and culturing in areprogramming media generates blood cell derived induced pluripotentstem cells. In other embodiments, delivering a quantity of reprogrammingfactors comprises nucleofection. In other embodiments, the reprogrammingfactors comprise one or more factors selected from the group consistingof: Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV40 Large T Antigen (“SV40LT”),and short hairpin RNAs targeting p53 (“shRNA-p53”). In otherembodiments, the reprogramming factors are encoded in one or moreoriP/EBNA1 derived vectors. In other embodiments, the one or moreoriP/EBNA1 derived vectors comprise pEP4 E02S ET2K,pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCXWB-EBNA1. In otherembodiments, plating of the blood cells on a treated cell culturesurface after delivering reprogramming factors into the blood cells, andculturing the blood cells in a reprogramming media on said treated cellculture surface. In other embodiments, the treated cell culture surfacecomprises plating of mouse embryonic feeders (MEFs). In otherembodiments, the treated cell culture surface comprises an extracellularmatrix protein. In other embodiments, the extracellular matrix proteincomprises laminin. In other embodiments, laminin comprises L-521. Inother embodiments, the reprogramming media comprises embryonic stem cell(ESC) media. In other embodiments, the ESC media comprises basicfibroblast growth factor (bFGF). In other embodiments, the reprogrammingmedia comprises E7 media. In other embodiments, the reprogramming mediacomprises E7 media comprising L-Ascorbic Acid, Transferrin, SodiumBicarbonate, Insulin, Sodium Selenite and/or bFGF. In other embodiments,culturing the blood cells in a reprogramming media is for at least 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days. In other embodiments,culturing the blood cells in a reprogramming media is for at least 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days. Furtherdescribed herein is a cell line comprising blood cell derived inducedpluripotent stem cells generated by the aforementioned method. In otherembodiments, the blood cells are isolated from a subject possessing adisease mutation. In other embodiments, the disease mutation isassociated with a neurodegenerative disease, disorder and/or condition.In other embodiments, the disease mutation is associated with aninflammatory bowel disease, disorder, and/or condition. In otherembodiments, the blood cells are non T-cell, non B-cell mononuclearcells. In other embodiments, the blood cells are a sample drawn from ahuman subject. In other embodiments, the sample is whole blood. In otherembodiments, the sample is peripheral blood. In other embodiments, thesample comprises an isolated component of non T-cell, non B-cellmononuclear cells. Further described herein is a blood cell derivedinduced pluripotent stem cell line.

Further described herein is a method for generating induced pluripotentstem cells, comprising providing a quantity of blood cells, delivering aquantity of reprogramming factors into the blood cells, plating theblood cells on a treated cell culture surface and culturing the bloodcells in a reprogramming media for at least 4 days, wherein deliveringthe reprogramming factors, and culturing in a reprogramming mediagenerates blood cell derived induced pluripotent stem cells. In otherembodiments, delivering a quantity of reprogramming factors comprisesnucleofection, the reprogramming factors comprise one or more factorsselected from the group consisting of: Oct-4, Sox-2, Klf-4, c-Myc,Lin-28, SV40 Large T Antigen (“SV40LT”), and short hairpin RNAstargeting p53 (“shRNA-p53”), encoded in one or more oriP/EBNA1 derivedvectors. In other embodiments, the one or more oriP/EBNA1 derivedvectors comprise pEP4 E02S ET2K, pCXLE-hOCT3/4-shp53-F, pCXLE-hSK,pCXLE-hUL, and pCXWB-EBNA1. In other embodiments, the treated cellculture surface comprises plating of mouse embryonic feeders (MEFs). Inother embodiments, the treated cell culture surface comprises anextracellular matrix protein. In other embodiments, the extracellularmatrix protein comprises laminin. In other embodiments, laminincomprises L-521. In other embodiments, the reprogramming media comprisesembryonic stem cell (ESC) media comprising basic fibroblast growthfactor (bFGF). In other embodiments, the reprogramming media comprisesE7 media comprising L-Ascorbic Acid, Transferrin, Sodium Bicarbonate,Insulin, Sodium Selenite and/or bFGF. In other embodiments, culturingthe blood cells in a reprogramming media is for at least 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 16 days. In other embodiments, culturingthe blood cells in a reprogramming media is for at least 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days. In other embodiments,the blood cells are a sample drawn from a human subject. In otherembodiments, the sample is whole blood. In other embodiments, the sampleis peripheral blood. In other embodiments, the sample comprises anisolated component of non T-cell, non B-cell mononuclear cells. Furtherdescribed herein is a blood cell derived induced pluripotent stem cellline.

BRIEF DESCRIPTION OF FIGURES

FIG. 1(A) to FIG. 1(B). Separation of Blood Components. FIG. 1(A)Schematic showing separated components. FIG. 1(B) Picture of separatedcomponents.

FIG. 2(A) to FIG. 2(E). Quality Control: iPS cells. Various measures ofquality control for generated cells are shown including FIG. 2(A)microscopy, and staining for pluripotent stem cell markers. FIG. 2(B)Depiction of the pluripotency of stem cells. FIG. 2(C) Karyotypeanalysis. FIG. 2(D) RT-PCR of pluripotent and lineage specific markersfor different cell lines. FIG. 2(E) Pluripotent stem cells as capable ofdifferentiating into all three embryonic lineages.

FIG. 3. PBMC isolation and cryopreservation process. Process forobtaining blood cells utilized as a source for reprogramming andgeneration of iPSCs.

FIG. 4. Peripheral blood mononuclear cells (PBMCs). Depiction of thevarious components of blood, including populations of PBMCs and relativepopulation abundance.

FIG. 5. T-cell Receptor Clonality Assay for iPSCs derived from Blood.

FIG. 6(A) to FIG. 6(B). Ideogram demonstrating the chromosomal changesobserved in the iPSC lines are Cedars-Sinai iPSC Core. Each colored barrepresents one chromosome change occurrence in one cell line. Thecytogenetic changes are color coded; Marooon, monosomy, loss of a wholechromosome. BC-iPSCs are superior at maintaining stable karyotypes thanfibroblast-derived iPSCs. Individual chromosome level ideogramrepresentation of abnormal karyotypes from fibroblast—(FIG. 6(A)) andPBMC-derived iPSCs (FIG. 6(B)) in our laboratory. Abnormal iPSCkaryotypes represented in the ideograms are; (FIG. 6(A)) fib-iPSCs (n:59) of total 258 fib-iPSC cultures karyotyped, and (FIG. 6(B)) BC-iPSCs(n: 4) of total BC-iPSCss karyotyped (n: 106).

FIG. 7(A) to 7(B). Karyotypes. FIG. 7(A) Distribution of abnormalkaryotypes in relation to passage number. FIG. 7(B) Distribution ofabnormal karyotypes in relation to age of tissue donor Marooon,monosomy, loss of a whole chromosome;

FIG. 8(A) to 8(F). Reliable epsiomal reprogramming of PBMCs into iPSCs.FIG. 8(A): Schematic depicting the episomal reprogramming process andtimeline of iPSC generation from PBMCs. FIG. 8(B): Bright-field imagesof the reprogrammed iPSC colonies from PBMCs obtained two healthyvolunteers, one with a non-T cell (03iCTR-NTn1) reprogramming method andanother with a T-cell (80iCTR-Tn1) reprogramming method, show a highnuclear-to-cytoplasmic ratio, are alkaline phosphatase positive andimmunopositive for the surface antigens, SSEA4, TRA-1-60, TRA-1-81; andnuclear pluripotency markers OCT3/4, SOX2 and NANOG. Scale bar, 75 μm.FIG. 8(C): Microarray Illumina gene-chip and bioinformatics basedPluriTest confirms pluripotency levels in the BC-iPSCs. (D): The PBMC--iPSC lines maintained a normal G-band karyotype as shown from the tworepresentative lines. FIG. 8(E): non-T or T-cell clonality assay. FIG.8(F): Quantitative RT-PCR analyses of POU5F1 (OCT4), SOX2, LIN28, L-MYC,and KLF4 expression in 49iCTR and 84iSMA LCL-iPSCs relative to H9 hESCs.(G) T cell receptor gene rearrangement assay shows different sets of PCRprimers were used to detect TCRG gene rearrangements occurring in theBC-iPSCs derived from the T-cell method, while PCR products in the145-255 bp range were not detectable in the non-T cell iPSC clones. Aclonal positive control was included. FIG. 8(F): TaqMan hPSC Scorecardtable showing the tri-lineage potential of the representative BC-iPSCsrom selected genes in four gene groups (self-renewal/pluripotency,ectoderm, endoderm, and mesoderm) comparing spontaneous in vitro EBdifferentiation of EBs derived from the PBMC-iPSC lines. FIG. 8(G):Directed neuronal differentiation of representative fibroblast-derived(n: 26) and PBMC-derived iPSCs (n: 12) showing equivalent numbers ofectodermal (Nkx6.1+/β₃-tubulin+neuronal cells). Scale bar, 75 μm.

FIG. 9(A) to FIG. 9(D): Compared to fib-iPSC, the BC-iPSCs are morestable over extended culture. FIG. 9(A) Percent abnormal G-bandkaryotypes observed in the iPSC lines in correlation to donor age offibroblasts and PBMCs. FIG. 9(B) Percent abnormal G-band karyotypes atthe first karyotype post-reprogramming and in repeat karyotypes for iPSClines evaluated in extended cell culture. The dotted lines represent theoverall average karyotype abnormality rate for fib-iPSCs (orange) andBC-iPSCs (blue). FIG. 9(C) Percent abnormal at the first G-bandkaryotypes evaluated for the fib- and PBMC-iPSC clonal lines in relationto the passage number of the iPSCs. FIG. 9(D) Percent abnormal at thesecond—fourth (repeat) G-band karyotypes evaluated for the fib- andPBMC-iPSC clonal lines in relation to the passage number of the iPSCs.

FIG. 10(A) to FIG. 10(E): Comparative genomic hybridization confirmsthat the BC-iPSCs have relatively small and fewer submicroscopiccytogenetic aberrations compared to fib-iPSCs. (FIG. 10(A)-FIG. 10(B))The average size of de novo acquired amplifications and deletionsdetected by aCGH in Mb of fib-iPSCs (n: 6) and PBMC-iPSCs (n:7) whencompared to each iPSC line's parental cells. The iPSC lines used in aCGHhad both abnormal and normal G-band karyotypes. Abnormal: fib-iPSC (n:3) and PBMC-iPSC (n: 2). Normal: fib-iPSC (n: 3) and PBMC-iPSC (n: 5).(FIG. 10(C)-FIG. 10(D)) The average number of de novo acquiredamplifications and deletions detected by aCGH in Mb of fib-iPSCs (n: 6)and PBMC-iPSCs (n: 7) when compared to each iPSC line's parental cells.FIG. 10(E) A list of most recurrent submicroscopic amps/dels observed inthe fib- or PBMC-iPSC lines observed in our laboratory.

FIG. 11(A) to FIG. 11(C): Transgene-free status of BC-iPSCs. FIG. 11(A)Lack of plasmid-based EBNA gene expression using a assay in the genomicDNA of the BC-iPSCs. FIG. 11 (B): Relative normalized gene expressionmeasured by quantitative RT-PCR analyses using primers detectingendogenous POU5F1 (OCT4), SOX2, LIN28, L-MYC, and KLF4 expression(coding DNA sequence, CDS). FIG. 11 (C) as well as plasmid-derivedexpression (Pla), which is undetectable in the iPSC lines.

FIG. 12(A) to FIG. 12(B): Chromosomal ideograms of lymphoblastoid cellline (LCL)-iPSCs and epithelial cell-derived iPSCs. Individualchromosome level ideogram representation of abnormal karyotypes fromLCL-derived FIG. 12(A) and epithelial-derived iPSCs FIG. 12(B) in ourlaboratory. Abnormal iPSC karyotypes represented in the ideograms are;(FIG. 12(A)) LCL-iPSCs (n: 15) of total 60 LCL-iPSC cultures karyotyped,and (FIG. 12(B)) epithelial-iPSCs (n: 7) of total BC-iPSCs karyotyped(n: 26).

DETAILED DESCRIPTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Allen et al., Remington: The Science and Practice of Pharmacy22^(nd) ed., Pharmaceutical Press (Sep. 15, 2012); Hornyak et al.,Introduction to Nanoscience and Nanotechnology, CRC Press (2008);Singleton and Sainsbury, Dictionary of Microbiology and MolecularBiology 3^(rd) ed., revised ed., J. Wiley & Sons (New York, N.Y. 2006);Smith, March's Advanced Organic Chemistry Reactions, Mechanisms andStructure 7^(th) ed., J. Wiley & Sons (New York, N.Y. 2013); Singleton,Dictionary of DNA and Genome Technology 3^(rd) ed., Wiley-Blackwell(Nov. 28, 2012); and Green and Sambrook, Molecular Cloning: A LaboratoryManual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor,N.Y. 2012), provide one skilled in the art with a general guide to manyof the terms used in the present application. For references on how toprepare antibodies, see Greenfield, Antibodies A Laboratory Manual2^(nd) ed., Cold Spring Harbor Press (Cold Spring Harbor N.Y., 2013);Köhler and Milstein, Derivation of specific antibody-producing tissueculture and tumor lines by cell fusion, Eur. J. Immunol. 1976 July,6(7):511-9; Queen and Selick, Humanized immunoglobulins, U.S. Pat. No.5,585,089 (1996 December); and Riechmann et al., Reshaping humanantibodies for therapy, Nature 1988 Mar. 24, 332(6162):323-7.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise.

Patient-specific induced pluripotent stem cells (“iPSCs”) hold greatpromise for many applications, including disease modeling to elucidatemechanisms involved in disease pathogenesis, drug screening, andultimately regenerative medicine therapies. A frequently used startingsource of cells for reprogramming has been dermal fibroblasts isolatedfrom skin biopsies. However, as described, iPSCs derived from skin punchbiopsies are more invasive, require a prolonged 2-3 week period ofexpansion in culture prior to reprogramming. Most importantly, there aregreater numbers of mutations associated with epidermal exposure to UVlight, raising concerns over the safety of the iPSCs cells derived fromskin. Issues with the common fibroblast cell source would becircumvented with peripheral blood, which can be utilized as an easilyaccessible source of patient tissue for reprogramming. Peripheral bloodis the most accessible adult tissue and permits access to numerousfrozen samples already stored at blood banks. Additionally, manyrepositories have stored peripheral or lymphoblast blood with specificgenotypes.

Maintaining genomic integrity and stability of hiPSC lines is imperativefor reliable disease modeling and safe clinical applications of stemcells as a regenerative therapy. Aberrant cytogenetic errors that ariseduring reprogramming of somatic cells and/or during maintenance,expansion and prolonged culture of hiPSCs will impact the accuracy of invitro disease modelling or, more critically, the in vivo utility ofiPSCs for regenerative medicine. It is important that iPSCs in clinicaluse are free from cancer-associated genomic aberrations, especiallygiven that several studies have reported chromosomal aneuploidy,translocations, duplications and deletions, and point mutations iniPSCs. The highly aneuploidy human embryonal carcinoma (EC) stem cells,which are the malignant analogues of normal hESCs, typically containamplified regions of the short arm of chromosome 12 and gains ofchromosomes 1, 17 and X.

Increased sub-chromosomal copy number variations (CNVs) with differencesin early, intermediate or late passage numbers, deletions associatedwith tumor-suppressor genes, and duplications of oncogenes have all beenreported in iPSCs In most of these studies, the genomic abnormalities inthe iPSC lines have been generated from integrating reprogrammingmethods. To date, however, no systematic and well-controlled (similarreprogramming and stem cell culture methods) reports of recurrentsub-chromosomal abnormalities specific to large numbers of fibroblast-or blood-derived hiPSC lines have been described. The Inventors havedeveloped an iPSC core facility with extensive experience producing,characterizing and differentiating human iPSC lines. This bio-repositorycurrently has over 160 well-characterized individual donor human iPSClines with multiple clones (2-6) per cell line. The iPSC Core hassystematically monitored the genomic stability of hiPSCs derived in theCore from 450 independent iPSC cultures over multiple passages, whichhave been derived from fibroblast, peripheral blood mononuclear cells,immortalized lymphoblastoid cell lines and primary epithelial cells.

Blood appears to an advantageous alternative for iPSC derivation aswidely used in clinical diagnostics, involving less invasive methods ofcollection that is standardized and less traumatic than skin biopsy. Themajor components of peripheral blood (PB) are red blood cells andplatelets. White blood cells are the nucleated cells in PB atconcentrations of 3.6−11×10⁶/ml. Mature T cells and primary progenitorcells in PB can be readily expanded using established methods and areamong the most successfully-used sources for reprogramming. T cells arethe most abundant cells after granulocytes in PB (20-30%) and T cellscan be readily expanded with IL-2 and anti-CD3/CD28 microbeads.Reprogramming of T cells into pluripotency has been reported by manylabs using different approaches for the sake of developing replacementsto aged T cells for immunotherapy. However, mature T cells harbor asingle T cell receptor (TCR) after somatic recombination and have lostthe ability to regenerate the T cell repertoire with unlimitedpossibilities.

Here the Inventors have developed a reliable protocol to efficientlyreprogram blood cells (BCs) including peripheral mononuclear blood cells(PBMCs) into iPSCs (BC-iPSCs) and show that these iPSC lines aresuperior in terms of cytogenetic stability in comparison to theirfibroblast-derived iPSC (Fib-iPSCs) lines obtained from publicrepositories or local clinics. The Inventors describe methods andcytogenetic stability for derivation of BC-iPSCs from both a lymphoid Tcell and a myeloid non-T cell population. The Inventors discovered thathiPSC lines generated from PBMCs using non-integrating methods havegreatly lower incidences of genomic aberrations than those generated byintegrating methods. Both fib-iPSCs and BC-iPSCs derived from the samesomatic cells contained comparable numbers of de novo CNVs. Our resultsshow that freshly isolated human PBMCs can be faithfully reprogrammed topluripotency with greater cytogenetic stability using episomal plasmids.As such, PBMCs should be a preferred somatic cell source for iPSCreprogramming to minimize any effects of acquired genomic aberrationsand, further, should be considered as an ideal cell source forregenerative medicine.

In contrast to mature T or B cells, the alternative source of bloodprogenitors contain an intact genome. In addition, they can be expandedin culture conditions that favor the proliferation of myeloid cells orerythroid cells. Blood stem/progenitor cells express surface marker CD34and reside in the stem cell niche. However, only about 1%stem/progenitor cells enter circulation each day and as a result, only0.01-0.1% cells in PB are CD34+ cells. This population can be enrichedby magnetic-activated cell sorting (MACS) or culture of MNCs for severaldays can be relied upon to expand CD34+ cells to a 5-20% purity, whichcan be used for reprogramming without further purification.

Other nucleated peripheral blood cells include granulocytes (mostlyneutrophils), monocytes, T lymphocytes, B lymphocytes and a fewprogenitor cells. Focusing on these constitutes of blood can be achievedby depleting red blood cells and platelet using lysis buffer followed bymultiple centrifugations. Ficoll gradient centrifugation can also beutilized to deplete both red blood cells and granulocytes, leading tothe enrichment of mononuclear cells (MNCs). Against this backdrop,reprogramming with exogenously expressed factors is notoriouslyinefficient and requires multiple cell cycles to achieve pluripotency.As such, primary granulocytes, monocytes and B lymphocytes are among themost difficult cells to be reprogrammed due to the lack of reliableprotocols to expand these cells. Primary success in this area includesEpstein-Barr virus immortalized lymphoblastoid B cells can be readilyexpanded in ex vivo culture and thus be reprogrammed to pluripotency.

In view of the above, of great interest is reprogramming of the non-Tcell component of blood. Existing techniques are largely unable toreprogram this population from isolated peripheral blood mononuclearcells (PBMCs). More specifically, PBMCs are any peripheral blood cellhaving a round nucleus. This includes lymphocytes (T cells, B cells, NKcells), monocytes, dendritic cells. Lymphocytes are Small (5-10 μm) andMedium (10-18 μm) and constitute 70-90% of PBMCs. Of these cells, 70-85%CD3+ T cells (40-70% of PBMCs), CD4 Helper T cells (25-60% of PBMCs),typically with CD4 to CD8 ratio of 2:1, CD8 “Cytotoxic” compartment Tcells (5-30% of PBMCs). The remaining compartment includes 5-20% B Cells(up to 15% of PBMCs) and 5-20% NK Cells (up to 15% of PBMCs). Monocytesare 16-25 μm and 10-30% of PBMCs (macrophages). Dendritic cells: 1-2% ofPBMCs

These described approaches allow for use of peripheral blood as areadily accessible resource for cellular reprogramming with superiorproperties in genomic and karyotype stability avoiding environmentalinsults for which mutations or other forms of structural alterationwould otherwise be therapeutic materials derived therein.

As described, the Inventors have established improved techniques forhighly efficient, reproducible reprogramming using non-integratingepisomal plasmid vectors, including generation of iPSCs from bloodcells, including whole blood and peripheral blood, the resultingreprogrammed pluripotent cells described herein as “BC-iPSCs”.

Generally, different approaches for non-integrative reprogramming spanat least categories: 1) integration-defective viral delivery, 2)episomal delivery, 3) direct RNA delivery, 4) direct protein deliveryand 5) chemical induction. As described further herein, the adoption ofepisomal vectors allows for generation of iPSCs substantially free ofthe vectors used in their production, as episomal or similar vectors donot encode sufficient viral genome sufficient to give rise to infectionor a replication-competent virus. At the same time, these vectors dopossess a limited degree of self-replication capacity in the beginningsomatic host cells. This self-replication capacity provides a degree ofpersistent expression understood to be beneficial in allowing thededifferentiation process to initiate take hold in a target host cell.

One example of a plasmid vector satisfying these criteria includes theEpstein Barr oriP/Nuclear Antigen-1 (“EBNA1”) combination, which iscapable of limited self-replication and known to function in mammaliancells. As containing two elements from Epstein-Barr virus, oriP andEBNA1, binding of the EBNA1 protein to the virus replicon region oriPmaintains a relatively long-term episomal presence of plasmids inmammalian cells. This particular feature of the oriP/EBNA1 vector makesit ideal for generation of integration-free iPSCs.

More specifically, persistent expression of reprogramming factor encodedin an oriP/EBNA1 vector occurs across multiple cell divisional cycles.Sufficiently high levels of reprogramming factors across several celldivisions allows for successful reprogramming even after only oneinfection. While sustained expression of reprogramming factors isunderstood to be beneficial during initial programming stages, otherwiseunlimited constitutive expression would hamper subsequent stages of thereprogramming process. For example, unabated expression of reprogrammingfactors would interfere with subsequent growth, development, and fatespecification of the host cells.

At the same time, a further benefit is the eventual removal of thereprogramming factor transgenes, as a small portion of episomes is lostper cell cycle. This is due to the asymmetric replication capacity ofthe host cell genome and episomal self-replication and it is estimatedthat approximately 0.5% of vector is lost per generation. Gradualdepletion of plasmids during each cell division is inevitable followingpropagation leading to a population of integration-free iPSCs. Thepersistent, yet eventual abrogation of reprogramming factor expressionon oriP/EBNA1 is highly coincident with the needs for different stagesof the reprogramming process and eliminates the need for furthermanipulation steps for excision of the reprogramming factors, as hasbeen attempted through use of transposons and excisable polycistroniclentiviral vector elements. Although oriP/EBNA1 has been applied byothers in reprogramming studies, the reported efficiencies are extremelylow (as few as 3 to 6 colonies per million cells nucleofected), whichmay be due, in-part, to reliance on large plasmids encoding multiplereprogramming factors (e.g., more than 12 kb), negatively impactingtransfection efficiency.

In addition to these choices in vector designs, the specificcombinations of reprogramming factors implemented in the literature havevaried. As mentioned, reprogramming factors that have been used includepluripotency-related genes Oct-4, Sox-2, Lin-28, Nanog, Sa114, Fbx-15and Utf-1. These factors, traditionally are understood be normallyexpressed early during development and are involved in the maintenanceof the pluripotent potential of a subset of cells that will constitutingthe inner cell mass of the pre-implantation embryo and post-implantationembryo proper. Their ectopic expression of is believed to allow theestablishment of an embryonic-like transcriptional cascade thatinitiates and propagates an otherwise dormant endogenous corepluripotency program within a host cell. Certain other reprogrammingdeterminants, such as Tert, Klf-4, c-Myc, SV40 Large T Antigen(“SV40LT”) and short hairpin RNAs targeting p53 (“shRNA-p53”) have beenapplied. There determinants may not be potency-determining factors inand of themselves, but have been reported to provide advantages inreprogramming. For example, TERT and SV40LT are understood to enhancecell proliferation to promote survival during reprogramming, whileothers such as short hairpin targeting of p53 inhibit or eliminatereprogramming barriers, such as senescence and apoptosis mechanisms. Ineach case, an increase in both the speed and efficiency of reprogrammingis observed. In addition, microRNAs (“miRNAs”) are also known toinfluence pluripotency and reprogramming, and some miRNAs from themiR-290 cluster have been applied in reprogramming studies. For example,the introduction of miR-291-3p, miR-294 or miR-295 into fibroblasts,along with pluripotency-related genes, has also been reported toincrease reprogramming efficiency.

While various vectors and reprogramming factors in the art appear topresent multiple ingredients capable of establishing reprogramming incells, a high degree of complexity occurs when taking into account thestoichiometric expression levels necessary for successful reprogrammingto take hold. For example, somatic cell reprogramming efficiency isreportedly fourfold higher when Oct-4 and Sox2 are encoded in a singletranscript on a single vector in a 1:1 ratio, in contrast to deliveringthe two factors on separate vectors. The latter case results in a lesscontrolled uptake ratio of the two factors, providing a negative impacton reprogramming efficiency. One approach towards addressing theseobstacles is the use of polycistronic vectors, such as inclusion of aninternal ribosome entry site (“IRES”), provided upstream of transgene(s)that is distal from the transcriptional promoter. This organizationallows one or more transgenes to be provided in a single reprogrammingvector, and various inducible or constitutive promoters can be combinedtogether as an expression cassette to impart a more granular level oftranscriptional control for the plurality of transgenes. These morespecific levels of control can benefit the reprogramming processconsiderably, and separate expression cassettes on a vector can bedesigned accordingly as under the control of separate promoters.

Although there are advantages to providing such factors via a single, orsmall number of vectors, upper size limitations on eventual vector sizedo exist, which can stymie attempts to promote their delivery in a hosttarget cell. For example, early reports on the use of polycistronicvectors were notable for extremely poor efficiency of reprogramming,sometimes occurring in less than 1% of cells, more typically less than0.1%. These obstacles are due, in-part, to certain target host cellspossessing poor tolerance for large constructs (e.g., fibroblasts), orinefficient processing of IRES sites by the host cells. Moreover,positioning of a factor in a vector expression cassette affects both itsstoichiometric and temporal expression, providing an additional variableimpacting reprogramming efficiency. Thus, some improved techniques canrely on multiple vectors each encoding one or more reprogramming factorsin various expression cassettes. Under these designs, alteration of theamount of a particular vector for delivery provides a coarse, butrelatively straightforward route for adjusting expression levels in atarget cell.

A further advantage of the techniques described herein is the use ofdefined media conditions for the reprogramming process, including theuse of ESC media and/or E7 media. While certain additives may be presentto spur the reprogramming process (e.g., L-Ascorbic Acid, Transferrin,Sodium Bicarbonate, Insulin, Sodium Selenite and/or bFGF), no serum oranimal components are used. In some instances, there may be furtherbenefits in altering the chemical and/or atmospheric conditions underwhich reprogramming will take place. For example, as thepre-implantation embryo is not vascularized and hypoxic (similar to bonemarrow stem-cell niches) reprogramming under hypoxic conditions of 5%O₂, instead of the atmospheric 21% O₂, may further provide anopportunity to increase the reprogramming efficiency. Similarly,chemical induction techniques have been used in combination withreprogramming, particularly histone deacetylase (HDAC) inhibitormolecule, valproic acid (VPA), which has been found wide use indifferent reprogramming studies. At the same time, other small moleculessuch as MAPK kinase (MEK)-ERK (“MEK”) inhibitor PD0325901, transforminggrowth factor beta (“TGF-β”) type I receptor ALK4, ALK5 and ALK7inhibitor SB431542 and the glycogen synthase kinase-3 (“GSK3”) inhibitorCHIR99021 have been applied for activation of differentiation-inducingpathways (e.g. BMP signaling), coupled with the modulation of otherpathways (e.g. inhibition of the MAPK kinase (MEK)-ERK pathway) in orderto sustain self-renewal. Other small molecules, such as Rho-associatedcoiled-coil-containing protein kinase (“ROCK”) inhibitors, such asY-27632 and thiazovivin (“Tzv”) have been applied in order to promotesurvival and reduce vulnerability of pSCs to cell death, particularlyupon single-cell dissociation.

In addition to the choice of delivery vectors, reprogramming factorcombinations, and conditions for reprogramming, further variations mustconsider the nature of the host target cell for reprogramming. To date,a wide variety of cells have served as sources for reprogrammingincluding fibroblasts, stomach and liver cell cultures, humankeratinocytes, adipose cells, and frozen human monocyte. Clearly, thereis a wide and robust potential for dedifferentiation across many tissuessources. Nevertheless, it is widely understood that depending on thedonor cell type, reprogramming is achieved with different efficienciesand kinetics. For example, although fibroblasts remain the most populardonor cell type for reprogramming studies, other types of cells such ashuman primary keratinocytes transduced with Oct-4, Sox-2, Klf-4 andc-Myc have been reported to reprogram 100 times more efficiently andtwo-fold faster. Additionally, some other cell types, such as cord bloodcells, may only require a subset of reprogramming factors, such as Oct-4and Sox-2 for dedifferentiation to take hold, while neural progenitorcells may only require Oct-4. Without being bound to any particulartheory, it is believed that differences in reprogramming efficiencyand/or reprogramming factor requirements of specific host cells resultfrom high endogenous levels of certain reprogramming factors and/orintrinsic epigenetic states that are more amenable to reprogramming.

Although these many other sources have been used across studies for thegeneration of iPSCs, mononuclear cells (MNCs) from peripheral blood (PB)are a highly attractive host cell candidate due to convenience andfeatures as an almost unlimited resource for cell reprogramming. PBcells in particular are relatively easy to isolate (e.g., blood draw)compared to isolation from other sources such as fibroblasts (e.g., skinbiopsy). These cells do not require laborious culturing and propagationprior to reprogramming, thereby reducing the overall time from whichreprogramming iPSCs can be obtained.

Following successful reprogramming, clonal selection allows forgeneration of pluripotent stem cell lines. Ideally, such cells possessrequisite morphology (i.e., compact colony, high nucleus to cytoplasmratio and prominent nucleolus), self-renewal capacity for unlimitedpropagation in culture (i.e., immortal), and with the capability todifferentiate into all three germ layers (e.g., endoderm, mesoderm andectoderm). Further techniques to characterize the pluripotency of agiven population of cells include injection into an immunocompromisedanimal, such as a severe combined immunodeficient (“SCID”) mouse, forformation of teratomas containing cells or tissues characteristic of allthree germ layers.

Described herein is a composition of blood cell derived inducedpluripotent stem cells (“BC-iPSCs”). In certain embodiments, thecomposition of blood cell derived induced pluripotent stem cellsincludes cells generated by providing a quantity of blood cells,delivering a quantity of reprogramming factors into the blood cells,culturing the blood cells in a reprogramming media for at least 4 days,wherein delivering the reprogramming factors, and culturing generatesthe blood cells derived induced pluripotent stem cells. In certainembodiments, the blood cells are T-cells. In other embodiments, theblood cells are non-T-cells. In other embodiments, the blood cells aremononuclear cells (MNCs), including for example peripheral bloodmononuclear cells (PBMCs). In other embodiments, the cells are primarygranulocytes, monocytes and B lymphocytes.

In certain embodiments, the reprogramming factors are Oct-4, Sox-2,Klf-4, c-Myc, Lin-28, SV40 Large T Antigen (“SV40LT”), and short hairpinRNAs targeting p53 (“shRNA-p53”). In other embodiments, thesereprogramming factors are encoded in a combination of vectors includingpEP4 E02S ET2K, pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL andpCXWB-EBNA1. This includes, for example, using about 0.5-1.0 ugpCXLE-hOCT3/4-shp53, 0.5-1.0 ug pCXLE-hSK, 0.5-1.0 ug pCXLE-UL, about0.25-0.75 ug pCXWB-EBNA1 and 0.5-1.0 ug pEP4 E02S ET2K. This includes,for example, using 0.83 ug pCXLE-hOCT3/4-shp53, 0.83 ug pCXLE-hSK, 0.83ug pCXLE-UL, 0.5 ug pCXWB-EBNA1 and 0.83 ug pEP4 E02S ET2K, wherein thestoichiometric ratio of SV40LT (encoded in pEP4 E02S ET2K) and EBNA-1(encoded in pCXWB-EBNA1) supports the reprogramming of non-T cellcomponent of blood, including peripheral blood mononuclear cells. Incertain other embodiments, the reprogramming media includes PD0325901,CHIR99021, HA-100, and A-83-01. In other embodiments, the culturing theblood cells in a reprogramming media is for 4-30 days. In variousembodiments, the blood cells are plated on a treated cell culturesurface after delivering a quantity of reprogramming factors. In variousembodiments, treatment includes plating of feeder cells, such as mouseembryonic fibroblasts. In other embodiments, treatment includes coatingwith extracellular matrix proteins. In various embodiment, extracellularmatrix proteins include laminin.

In various embodiments, the BC-iPSCs are capable of serial passaging asa cell line. In various embodiments, the BC-iPSCs possess genomicstability. Genomic stability can be ascertained by various techniquesknown in the art. For example, G-band karyotyping can identify abnormalcells lacking genomic stability, wherein abnormal cells possess about10% or more mosaicism, or one or more balanced translocations of greaterthan about 5, 6, 7, 8, 9, 10 or more Mb. Alternatively, genomicstability can be measured using comparative genomic hybridization (aCGH)microarray, comparing for example, BC-iPSCs against iPSCs from anon-blood cell source such as fibroblasts. Genomic stability can includecopy number variants (CNVs), duplications/deletions, and unbalancedtranslocations. In various embodiments, BC-iPSCs exhibit no more thanabout 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, or 20 Mbaverage size of amplification and deletion. In various embodiments,BC-iPSCs exhibit no more than about 20-30 Mb average size ofamplification and deletion. In various embodiments, BC-iPSCs exhibit nomore than about 30-40 Mb average size of amplification and deletion. Invarious embodiments, BC-iPSCs exhibit no more than about 40-50 Mbaverage size of amplification and deletion. In various embodiments, theaverage number of acquired de novo amplification and deletions inBC-iPSCs is less than about 5, 4, 3, 2, or 1. For example, de novoamplification and deletions in fib-iPSCs are at least two-fold greaterthan in PBMC-iPSCs.

In different embodiments, reprogramming factors can also include one ormore of following: Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV40LT,shRNA-p53, nanog, Sa114, Fbx-15, Utf-1, Tert, or a Mir-290 clustermicroRNA such as miR-291-3p, miR-294 or miR-295. In differentembodiments, the reprogramming factors are encoded by a vector. Indifferent embodiments, the vector can be, for example, a non-integratingepisomal vector, minicircle vector, plasmid, retrovirus (integrating andnon-integrating) and/or other genetic elements known to one of ordinaryskill. In different embodiments, the reprogramming factors are encodedby one or more oriP/EBNA1 derived vectors. In different embodiments, thevector encodes one or more reprogramming factors, and combinations ofvectors can be used together to deliver one or more of Oct-4, Sox-2,Klf-4, c-Myc, Lin-28, SV40LT, shRNA-p53, nanog, Sa114, Fbx-15, Utf-1,Tert, or a Mir-290 cluster microRNA such as miR-291-3p, miR-294 ormiR-295. For example, oriP/EBNA1 is an episomal vector that can encode avector combination of multiple reprogramming factors, such as pCXLE-hUL,pCXLE-hSK, pCXLE-hOCT3/4-shp53-F, pEP4 EO2S T2K and pCXWB-EBNA1.

In other embodiments, the reprogramming factors are delivered bytechniques known in the art, such as nuclefection, transfection,transduction, electrofusion, electroporation, microinjection, cellfusion, among others. In other embodiments, the reprogramming factorsare provided as RNA, linear DNA, peptides or proteins, or a cellularextract of a pluripotent stem cell.

In various embodiments, the reprogramming media is embryonic stem cell(ESC) media. In various embodiments, the reprogramming media includesbFGF. In various embodiments, the reprogramming media is E7 media. Invarious embodiments, the reprogramming E7 media includes L-AscorbicAcid, Transferrin, Sodium Bicarbonate, Insulin, Sodium Selenite and/orbFGF In different embodiments, the reprogramming media comprises atleast one small chemical induction molecule. In different embodiments,the at least one small chemical induction molecule comprises PD0325901,CHIR99021, HA-100, A-83-01, valproic acid (VPA), SB431542, Y-27632 orthiazovivin (“Tzv”). In different embodiments, culturing the BCs in areprogramming media is for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30days.

In certain embodiments, the BC-iPSCs are derived from blood cellspreviously isolated from a subject, by for, example, drawing a bloodsample from the subject. In other embodiments, the blood cells areisolated from a subject possessing a disease mutation. For example,subjects possessing any number of mutations, such as autosomal dominant,recessive, sex-linked, can serve as a source of blood cells to generateBC-iPSCs possessing said mutation. In other embodiments, the diseasemutation is associated with a neurodegenerative disease, disorder and/orcondition. In other embodiments, the disease mutation is associated withan inflammatory bowel disease, disorder, and/or condition. In variousembodiments, the BC-iPSCs possess features of pluripotent stem cells.Some exemplary features of pluripotent stem cells includingdifferentiation into cells of all three germ layers (ectoderm, endoderm,mesoderm), either in vitro or in vivo when injected into animmunodeficient animal, expression of pluripotency markers such asOct-4, Sox-2, nanog, TRA-1-60, TRA-1-81, SSEA4, high levels of alkalinephosphatase (“AP”) expression, indefinite propagation in culture, amongother features recognized and appreciated by one of ordinary skill.

Other embodiments include a pharmaceutical composition including aquantity of blood cells derived induced pluripotent stem cells generatedby the above described methods, and a pharmaceutically acceptablecarrier.

Also described herein is an efficient method for generating inducedpluripotent stem cells, including providing a quantity of cells,delivering a quantity of reprogramming factors into the cells, culturingthe cells in a reprogramming media for at least 4 days, whereindelivering the reprogramming factors, and culturing generates inducedpluripotent stem cells. In certain embodiments, the cells are primaryculture cells. In other embodiments, the cells are blood cells (BCs). Incertain embodiments, the blood cells are T-cells. In other embodiments,the blood cells are non-T-cells. In other embodiments, the cells aremononuclear cells (MNCs), including for example peripheral bloodmononuclear cells (PBMCs). In other embodiments, the cells are primarygranulocytes, monocytes and B lymphocytes.

In certain embodiments, the reprogramming factors are Oct-4, Sox-2,Klf-4, c-Myc, Lin-28, SV40 Large T Antigen (“SV40LT”), and short hairpinRNAs targeting p53 (“shRNA-p53”). In other embodiments, thesereprogramming factors are encoded in a combination of vectors includingpEP4 E02S ET2K, pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL andpCXWB-EBNA1. This includes, for example, using about 0.5-1.0 ugpCXLE-hOCT3/4-shp53, 0.5-1.0 ug pCXLE-hSK, 0.5-1.0 ug pCXLE-UL, about0.25-0.75 ug pCXWB-EBNA1 and 0.5-1.0 ug pEP4 E02S ET2K. This includes,for example, using 0.83 ug pCXLE-hOCT3/4-shp53, 0.83 ug pCXLE-hSK, 0.83ug pCXLE-UL, 0.5 ug pCXWB-EBNA1 and 0.83 ug pEP4 E02S ET2K, wherein thestoichiometric ratio of SV40LT (encoded in pEP4 E02S ET2K) and EBNA-1(encoded in pCXWB-EBNA1) supports the reprogramming of non-T cellcomponent of blood, including peripheral blood mononuclear cells. Invarious embodiments, the reprogramming media is embryonic stem cell(ESC) media. In various embodiments, the reprogramming media includesbFGF. In various embodiments, the reprogramming media is E7 media. Invarious embodiments, the reprogramming E7 media includes L-AscorbicAcid, Transferrin, Sodium Bicarbonate, Insulin, Sodium Selenite and/orbFGF. In different embodiments, the reprogramming media comprises atleast one small chemical induction molecule. In certain otherembodiments, the reprogramming media includes PD0325901, CHIR99021,HA-100, and A-83-01. In other embodiments, the culturing the blood cellsin a reprogramming media is for 4-30 days.

In various embodiments, the BC-iPSCs are capable of serial passaging asa cell line. In various embodiments, the BC-iPSCs possess genomicstability. Genomic stability can be ascertained by various techniquesknown in the art. For example, G-band karyotyping can identify abnormalcells lacking genomic stability, wherein abnormal cells possess about10% or more mosaicism, or one or more balanced translocations of greaterthan about 5, 6, 7, 8, 9, 10 or more Mb. Alternatively, genomicstability can be measured using comparative genomic hybridization (aCGH)microarray, comparing for example, BC-iPSCs against iPSCs from anon-blood cell source such as fibroblasts. Genomic stability can includecopy number variants (CNVs), duplications/deletions, and unbalancedtranslocations. In various embodiments, BC-iPSCs exhibit no more thanabout 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, or 20 Mbaverage size of amplification and deletion. In various embodiments,BC-iPSCs exhibit no more than about 20-30 Mb average size ofamplification and deletion. In various embodiments, BC-iPSCs exhibit nomore than about 30-40 Mb average size of amplification and deletion. Invarious embodiments, BC-iPSCs exhibit no more than about 40-50 Mbaverage size of amplification and deletion. In various embodiments, theaverage number of acquired de novo amplification and deletions inBC-iPSCs is less than about 5, 4, 3, 2, or 1. For example, de novoamplification and deletions in fib-iPSCs are at least two-fold greaterthan in PBMC-iPSCs. In various embodiments, the methods produces iPSCcell lines collectively exhibiting about 20%, 15%, 10%, 5% or lessabnormal karyotypes over 4-8, 9-13, 13-17, 17-21, 21-25, or 29 or morepassages when serially passaged as a cell line.

In different embodiments, reprogramming factors can also include one ormore of following: Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV40LT,shRNA-p53, nanog, Sa114, Fbx-15, Utf-1, Tert, or a Mir-290 clustermicroRNA such as miR-291-3p, miR-294 or miR-295. In differentembodiments, the reprogramming factors are encoded by a vector. Indifferent embodiments, the vector can be, for example, a non-integratingepisomal vector, minicircle vector, plasmid, retrovirus (integrating andnon-integrating) and/or other genetic elements known to one of ordinaryskill. In different embodiments, the reprogramming factors are encodedby one or more oriP/EBNA1 derived vectors. In different embodiments, thevector encodes one or more reprogramming factors, and combinations ofvectors can be used together to deliver one or more of Oct-4, Sox-2,Klf-4, c-Myc, Lin-28, SV40LT, shRNA-p53, nanog, Sa114, Fbx-15, Utf-1,Tert, or a Mir-290 cluster microRNA such as miR-291-3p, miR-294 ormiR-295. For example, oriP/EBNA1 is an episomal vector that can encode avector combination of multiple reprogramming factors, such as pCXLE-hUL,pCXLE-hSK, pCXLE-hOCT3/4-shp53-F, pEP4 EO2S T2K and pCXWB-EBNA1.

In other embodiments, the reprogramming factors are delivered bytechniques known in the art, such as nuclefection, transfection,transduction, electrofusion, electroporation, microinjection, cellfusion, among others. In other embodiments, the reprogramming factorsare provided as RNA, linear DNA, peptides or proteins, or a cellularextract of a pluripotent stem cell. In certain embodiments, the cellsare treated with sodium butyrate prior to delivery of the reprogrammingfactors. In other embodiments, the cells are incubated or 1, 2, 3, 4, ormore days on a tissue culture surface before further culturing. This caninclude, for example, incubation on a Matrigel coated tissue culturesurface. In other embodiments, the reprogramming conditions includeapplication of norm-oxygen conditions, such as 5% O₂, which is less thanatmospheric 21% O₂.

In various embodiments, the reprogramming media is embryonic stem cell(ESC) media. In various embodiments, the reprogramming media includesbFGF. In various embodiments, the reprogramming media is E7 media. Invarious embodiments, the reprogramming E7 media includes L-AscorbicAcid, Transferrin, Sodium Bicarbonate, Insulin, Sodium Selenite and/orbFGF. In different embodiments, the reprogramming media comprises atleast one small chemical induction molecule. In different embodiments,the at least one small chemical induction molecule comprises PD0325901,CHIR99021, HA-100, A-83-01, valproic acid (VPA), SB431542, Y-27632 orthiazovivin (“Tzv”). In different embodiments, culturing the BCs in areprogramming media is for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30days.

Efficiency of reprogramming is readily ascertained by one of manytechniques readily understood by one of ordinary skill. For example,efficiency can be described by the ratio between the number of donorcells receiving the full set of reprogramming factors and the number ofreprogrammed colonies generated. Measuring the number donor cellsreceiving reprogramming factors can be measured directly, when areporter gene such as GFP is included in a vector encoding areprogramming factor. Alternatively, indirect measurement of deliveryefficiency can be provided by transfecting a vector encoding a reportergene as a proxy to gauge delivery efficiency in paired samplesdelivering reprogramming factor vectors. Further, the number ofreprogrammed colonies generated can be measured by, for example,observing the appearance of one or more embryonic stem cell-likepluripotency characteristics such as alkaline phosphatase (AP)-positiveclones, colonies with endogenous expression of transcription factors Octor Nanog, or antibody staining of surface markers such as Tra-1-60. Inanother example, efficiency can be described by the kinetics of inducedpluripotent stem cell generation. For example, efficiency can includeproducing cell lines of normal karyotype, including the method producingiPSC cell lines collectively exhibiting about 20%, 15%, 10%, 5% or lessabnormal karyotypes over 4-8, 9-13, 13-17, 17-21, 21-25, or 29 or morepassages when serially passaged as a cell line.

Example 1 General iPSC Reprogramming Protocol for Blood

Generally, the reprogramming method involves the following steps. Frozenperipheral blood mononuclear cells (PBMCs), or freshly isolated PBMCscan be used. Treated cell culture surfaces can be used for platingthawed or freshly isolated PBMS. This includes, for example, cellculture surfaces treated with mouse embryonic feeders (MEF) orextracellular matrix laminin L-521A. An expression plasmid mixture isprovided, with the plasmid mixture encoding a combination ofpluripotency factors. Plasmids are introduced to PBMCs, by using forexample, nucleofection, and thereafter placed on the treated cellculture surface. After 2 days of culturing, reprogramming media isintroduced, and replenished until colonies are formed.

Example 2 Peripheral Blood Collection and Minimal Processing forReprogramming

The Inventors have established processes to isolate lymphocytes fromfreshly collected or commercial sources of human or mammalian peripheralblood (PB). The preferred format for the collection and shipment of suchsamples is the CPT tubes. This format allows the supplier to centrifugethe vacutainer(s) and separate the red blood cells from the plasmacomponents prior to shipping. The protocol provides for the isolation ofPBMCs from the plasma layer of the CPT tubes.

Optional tests: Prior to opening the Vacutainer, several disease statetests can be performed, including testing for HIV, Hepatitis B and C,and syphilis. The latter test results should be made available within1-3 working days from the collection date. If there is a positiveresult, the sample(s) will be properly discarded, and all laboratoryequipment that was used to process the samples will be decontaminated.

Example 3 Collection Procedure

(1) Red blood cells and platelets are depleted using containers. BDVacutainer® CPT™ Tube with Sodium Citrate should be at room temperature(18-25° C.) and properly labeled for patient identification. Spray theCPT Vacutainer lightly with 70% isopropanol/ethanol, wipe the alcoholoff.

(2) Blood collection is into three (3) 8 ml CPT tubes per sample usingthe standard technique for BD Vacutainer® Evacuated Blood CollectionTubes. After collection, tube is stored upright at room temperatureuntil centrifugation. Blood samples should be centrifuged within twohours of blood collection for best results.

(3) Tube/blood sample is centrifuged at room temperature (18-25° C.) ina horizontal rotor (swing-out head) for a minimum of 20 minutes (up to30 minutes) at 1500 to 1800 RCF (Relative Centrifugal Force).

(4) For blood shipped in CPT tubes and reprogramming is performed within24 hours: After centrifugation, mononuclear cells and platelets will bein a whitish layer just under the plasma layer (see FIG. 1). Analternative procedure for recovering the separated mononuclear cells isto resuspend the cells into the plasma by inverting the unopened BDVacutainer® CPT™ Tube gently 5 to 10 times. This is the preferred methodfor storing or transporting the separated sample for up to 24 hoursafter centrifugation.

Example 4 Important Parameters

Temperature: Since the principle of separation depends on a densitygradient, and the density of the components varies with temperature, thetemperature of the system should be controlled between 18-25° C. duringseparation.

Centrifugation: Since the principle of separation depends on themovement of formed elements in the blood through the separation media,the “RCF” should be controlled at 1500 RCF to 1800 RCF. The time ofcentrifugation should be a minimum of 20 minutes. (As noted in thetrouble shooting section, some specimens may require up to 30 minutesfor optimal separation.) Centrifugation of the BD Vacutainer® CPT™ Tubeup to 30 minutes has the effect of reducing red blood cell contaminationof the mononuclear cell fraction. Centrifugation beyond 30 minutes haslittle additional effect. The BD Vacutainer® CPT™ Tube may bere-centrifuged if the mononuclear “band” or layer is not disturbed.

Time: Blood samples should be centrifuged or separated within two hoursof blood drawing. Red blood cell contamination in the separatedmononuclear cell fraction increases with longer delays in sampleseparation. Mononuclear cell recovery decreases with increased timedelay before centrifugation, falling to approximately 40% mononuclearcell recovery at 24 hours. Pour the contents (the plasma layer) off intoan appropriately labeled 50 mL tube.

Example 5 Reprogramming Procedure Materials and Supplies

Described herein is the procedure for reprogramming separated wholeblood samples (peripheral blood mononuclear cells—PBMCs) for iPSCgeneration. Materials for use include: Sterile 1.5 ml Eppendorf tubes;Sterile pipette tips (1000 ul, 200 ul, 10 ul); Amaxa Nucleofector™ 2bDevice (Lonza); Human T-Cell Nucleofector Kit (Lonza, Cat #VPA-1002);Prepared MEF 6-well TC Plates or L-521 Coated Plates; 0.22 um SteriFlip(optional); Primate ESC Medium or E7 Medium, described below in Tables1-4.

Reprogramming Plasmids used include (1) pCXLE-hOCT3/4-shp53; (2)pCXLE-hSK; (3) pCXLE-UL; (4) pCXWB-EBNA1; (5) pEP4 E02S ET2K.

TABLE 1 αβ T-Cell Media ITEM CONCENTRATION X-vivo10 IL-2 30 U/mlDynabeads Human T-activator 5 ul/well CD3/CD28 (to be added afterNucleofection)

TABLE 2 Non-T Cell Media ITEM CONCENTRATION αMEM FBS 10% IL-3 10 ng/mlIL-6 10 ng/ml G-CSF 10 ng/ml GM-CSF 10 ng/ml

TABLE 3 MEF Media (optional) ITEM CONCENTRATION DMEM FBS 10% NEAA  1%GlutaMax  1%

TABLE 4 Prepared Primate ESC Medium (Optional) ITEM CONCENTRATIONPrimate ESC Medium bFGF 5 ng/ml

TABLE 5 Prepared E7 Medium (Optional) ITEM CONCENTRATION DMEM/F12L-Ascorbic Acid 64 ug/ml Transferrin 10.7 ug/ml Sodium Bicarbonate 543ug/ml Insulin 19.4 ug/ml Sodium Selenite 14 ng/ml bFGF 100 ng/ml

Example 6 Reprogramming Procedure

Day-1—Preparation of Plates

MEF Plates—

1. Coat each well of 6-well plate with 1 ml of 0.1% Gelatin. 2. Incubateplate at 37° C. for a minimum of 1 hour. 3. Obtain a vial of frozen MEFsfrom the LN2 tank. 4. Thaw vial in a water bath by gently moving thefrozen vial in a FIG. 8 motion in the water. 5. Collect thaw cells intoa 15 ml conical. 6. Slowly add MEF media to bring the volume to 10 mls.7. Centrifuge tube/MEFs at room temperature (18-25° C.) at 1000 to 1200RPM (Relative Centrifugal Force) for 5 minutes. 8. While the cells arespinning, aspirate the gelatin from the 6-well plate and add 1 ml of MEFmedia to each well. 9. After the centrifuged has stopped, remove theconical and aspirate the supernatant without disturbing the cell pellet.10. Resuspend the cell pellet in enough MEF media to achieveapproximately 50,000 cell s/ml. 11. Add 1 ml of cell/MEF media mixtureto each well. 12. Swirl plate to ensure even distribution of MEFs andplace in incubator overnight. 13. Prepare 10 ug/ml of L-521 by thawingone 1 ml vial of 100 ug/ml L-521 and adding the contents to 9 mls ofsterile PBS. 14. Filter the mixture through a 0.22 um filter orSteriFlip. 15. Add 1 ml of the 10 ug/ml L-521 mixture to each well of a6-well plate. 16. Wrap the plate with Parafilm and place in the 37° C.incubator for 2 hours. 17. Remove plate from the incubator and place in4° C. fridge.

Day 0—Cell Preparation of Frozen PBMCs

18. Thaw 2 frozen vials of PBMCs containing 5e6 cells each in the waterbath using a “FIG. 8” motion until a small ball of ice remains. 19.Collect the contents of each tube into two separate sterile 15 mlconicals and label the tubes 1 and 2. 20. Add sterile PBS to each tubeto bring the volume to 10 mls. Mix cells by inverting tube 5 times. 21.Centrifuge for 15 minutes at 1000 RPM. 22. Proceed to step 25

Cell Preparation of Freshly Isolated PBMCs

23. Place 3e6 freshly isolated PBMCs into 2 sterile 15 ml conicals each.24. Perform steps 19 to 22.

Reprogramming

If using L-521 coated plates, one allows plates to equilibrate toroom-temperature for at least 1 hr. 25. Prepare a 1.5 ml Eppendorf tubesof Lonza Nucleofection solution according to protocol (82 ul ofNucleofection Solution+18 ul of Supplement per reaction). Label the tubeNS.

NOTE: One will be performing each reaction twice; once for T-cellconditions and once for non T-cell conditions. It is okay to prepare amaster mix of NS for all reactions.

26. In an 1.5 ml Eppendorf tube, prepare 3.82 ug of expression plasmidmixture for each reaction as follows:

-   -   i. pCXLE-hOCT3/4-shp53: 0.83 ug    -   ii. pCXLE-hSK: 0.83 ug    -   iii. pCXLE-UL: 0.83 ug    -   iv. pCXWB-EBNA1: 0.5 ug    -   v. pEP4 E02S ET2K: 0.83 ug

27. Prepare a 15 ml conical with 6 mls T-cell media. 28. Prepare a 15 mlconical with 6 mls non T-cell media. 29. Aspirate the supernatant fromone of the 15 ml conicals containing your cells. 30. Gently flick thetube to loosen up the cell pellet. 31. Add 100 ul of NS mixture to yourcell pellet and gently pipette up and down 3-4 times. 32. Transfer thetotal volume of NS/cell mixture to a 1.5 ml Eppendorf tube containingyour plasmid mixture and mix by gently pipetting up and down 3-4 times.33. Transfer the total volume of NS/cell/plasmid mixture to a glasscuvette provided with the Lonza T-cell kit. 34. Place the cuvette in theAmaxa nucleofector and run program V-024. 35. Using the plastic pipettetip provided with the Lonza T-cell kit, transfer a small amount ofT-cell media to the glass cuvette then transfer the entire contents ofthe cuvette to the 15 ml conical containing 6 mls of T-cell media. 36Repeat steps 29-34 for your second 15 ml conical containing cells,proceed to step 37.

37. Using the plastic pipette tip provided with the Lonza T-cell kit,transfer a small amount of non T-cell media to the glass cuvette thentransfer the entire contents of the cuvette to the 15 ml conicalcontaining 6 mls of non T-cell media.

Plate Down

MEF/Primate ESC Condition

38. Remove the MEF media from each of the wells. 39. Rinse each wellwith 1 ml of sterile PBS. 40. Transfer the entire contents of the 15 mlconical from step 35 to the top 3 wells by adding 2 mls to each well.41. Add 5 ul of Dynabeads Human T-activator CD3/CD28 to each well. 42.Transfer the entire contents of the 15 ml conical from step 37 to thebottom 3 wells by adding 2 mls to each well. 43. Place the plate in a37° C. incubator.

L-521/E7 Condition

44. Aspirate the L-521 from each well. 45. Transfer the entire contentsof the 15 ml conical from step 5.35 to the top 3 wells by adding 2 mlsto each well. 46. Add 5 ul of Dynabeads Human T-activator CD3/CD28 toeach well. 47. Transfer the entire contents of the 15 ml conical fromstep 37 to the bottom 3 wells by adding 2 mls to each well. 48. Placethe plate in a 37° C. incubator.

Day 2

49. On day 2 post nucleofection, add 2 mls of the appropriatereprogramming media (Primate ESC+bFGF or E7) to each well. DO NOTASPIRATE ANYTHING FROM THE WELLS.

Day 4-25+

50. Beginning on day 4, gently aspirate the media from each well and add2 mls fresh reprogramming media to each well. This will be done everyother day.

Day 25+

51. Using a pulled glass pipette, isolate individual colonies andtransfer single colonies into 1 well of 12-well dish containing theappropriate substrate.

Example 7

A variety of quality control assays can be performed to confirm propergeneration of pluripotent stem cells. Examples of such testsexemplifying desirable characteristics of pSC generation are presentedin Table 1.

TABLE 1 iPSC QC Assays Test Assay/Kit Result Mycoplasma TestingMycoAlert-Lonza Negative Alkaline Phosphatase Alkaline Positive (AP)Staining Phosphatase Staining Kit II-Stemgent Immunocytochemistry ICCStaining Positive (ICC) for Pluripotency Markers-Oct, SSEA4, Nanog,Tra-1-60, Tra-1-81 Endogenous pluripotency qPCR Positive gene expressiongenes turned on-OCT4, SOX2, LIN28, KLF4, L-myc Lack of exogenous geneqPCR Negative gene expression presence G-Band Karyotype N/A Normal HumanKaryotype Illumina gene-chip Pluritest Pluripotency score >20,expression and Novelty score <1.6 bioinformatics assay EB Formation andTri- TaqMan_hPSC Positive for Endoderm, Ectoderm and lineage geneexpression Scorecard-Life Mesoderm Technologies Short tandem repeat(STR) Cell Check 9- Genetic profile of the sample matches profile andinterspecies IDEXX identically to the parental genetic profile.contamination testing Samples are confirmed to be of human origin and nomammalian interspecies contamination is detected. T-Cell Clonality AssayTCRB + TCRG T- Presence (T-cell) of lack of clonal (for whole bloodderived Cell Clonality TCR beta gene rearrangements iPSC lines) Assay ™Gel (non T-cell) Detection-Invivoscribe

Example 8 Alkaline Phosphatase Staining

For characterization of BC-iPSCs, Alkaline Phosphatase staining can beperformed using the Alkaline Phosphatase Staining Kit II (Stemgent, Catno. 00-0055) according to the manufacturer's instructions.

Example 9 Immunohisto/Cytochemistry

For further characterization of BC-iPSCs, BC-iPSCs or differentiatedcells can be plated on glass coverslips or optical-bottom 96-well plates(Thermo, #165305) and subsequently fixed in 4% paraformaldehyde.Intestinal organoids can be fixed in 4% paraformaldehyde, transferred to30% sucrose, embedded in HistoPrep (Thermo Fisher Scientific) and cutinto 20 μm sections. All cells are blocked in 510% goat or donkey serumwith 0.1% Triton X-100 and incubated with primary antibodies either foreither 3 hrs at room temperature or overnight at 4° C. Cells are thenrinsed and incubated in species-specific AF488 or AF594-conjugatedsecondary antibodies followed by Hoechst 33258 (0.5 μg/ml; Sigma) tocounterstain nuclei.

Antibodies suitable for immunocytochemistry and immunoblotting include:(as listed by antigen, dilution, catalog no., isotype, and manufacturer)SSEA4, 1:250, MAB4304, mIgG3, Millipore; TRA-1-60, 1:250, 09-0010, IgM,Stemgent; TRA-1-81, 1:250, 09-0011, mIgM, Stemgent; OCT4, 1:250,09-0023, Rabbit IgG, Stemgent; NANOG, 1:250, 09-0020, Rabbit IgG,Stemgent; SOX2,1:500, AB5603, Rabbit IgG, Millipore; Tull (β3-tubulin),1:1000, T8535, mIgG2b, Sigma; CDX2, 1:500, NBP1-40553, IgG, Novus;FABP2, 1:500, AF3078, IgG, R & D systems; Collagen Type 1, 1:500,600-401-103-0.1, Rabbit Rockland; CD73, 1:500, 550257, mIgG-1, BDPharmingen; NKX6.1, 1:100 F55A10, mIgG1, DSHB Iowa; HB9, 1:25, 81.5C10,mIgG1, DSHB Iowa; ISELT1, 1:250, AF1837, Goat IgG, R & D systems; SMI32,1:1000, SMI-32R, mIgG1, Covance; CHAT, 1:250, AB144P, Goat IgG,Millipore; SMN, 1:250, 610647, mIgG1, BD Biosciences; Cox-IV, 1:1000,4850s, Rabbit Cell signaling; GAPDH, 1:1000, ab9484, mIgG2b, Abcam.

Example 10 Flow Cytometry

Additional characterization of can include flow cytometry. Specifically,BC-iPSCs are dissociated into a single cell suspension using Accutase(Millipore, #SCR005). Surface staining of IPSCs is carried out usingSSEA4 (R&D Systems, FAB1435A).

Cells were then fixed, permeabilized and stained intracellularly forOct3/4 (BD Pharmingen, 560186). Recommended isotypes are used accordingto the antibodies recommendation (R&D Systems, FABIC007 and BDPharmingen, 562547). All samples re analyzed using a BD LSRFortessa flowcytometer using BD FACSDiva software. All images are generated usingFloJo software.

Example 11 Karyotype

Karyotyping can also be performed as follows. Human BC-iPSCs areincubated in Colcemid (100 ng/mL; Life Technologies) for 30 minutes at37° C. and dissociated using TrypLE for 10 minutes. They can be washedin phosphate buffered saline (PBS) and incubated at 37° C. in 5 mLhypotonic solution (1 g KCl, 1 g Na Citrate in 400 mL water) for 30minutes. The cells can be centrifuged for 2.5 minutes at 1500 RPM andresuspended in fixative (methanol: acetic acid, 3:1) at room temperaturefor 5 minutes. This is repeated twice, and finally cells wereresuspended in 500 μl of fixative solution and submitted to theCedars-Sinai Clinical Cytogenetics Core for G-Band karyotyping.

Example 12 PluriTest

Pluritest provides a robust measurement of pluripotency. Total RNA canbe isolated using the RNeasy Mini Kit (Qiagen) and subsequently run on aHuman HT-12 v4 Expression BeadChip Kit (Illumina). The raw data file(idat file) was subsequently uploaded onto the Pluritest widget online(www.pluritest.org).

Example 13 Quantitative RT-PCR

Total RNA was isolated using the RNeasy Mini Kit (Qiagen), and 1 ug ofRNA was used to make cDNA using the transcription system (Promega).qRT-PCR was performed using specific primer sequences (Table 2) understandard conditions. “CDS” indicates that primers designed for thecoding sequence measured expression of the total endogenous geneexpression only, whereas “Pla” indicates that primers designed for theplasmid transgene expression only. Data are represented as mean±SEM

TABLE 2  qRT-PCR Primer Sequences Gene Name Forward PrimerReverse Primer OCT3/4 CDS ccccagggccccattttggtaccacctcagtttgaatgcatgggagagc (SEQ ID NO: 1) (SEQ ID NO: 2) OCT3/4 Placattcaaactgaggtaaggg tagcgtaaaaggagcaacatag (SEQ ID NO: 3)(SEQ ID NO: 4) SOX2 CDS ttcacatgtcccagcactaccagatcacatgtgtgagaggggcagtgtgc (SEQ ID NO: 5) (SEQ ID NO: 6) SOX2 Plattcacatgtcccagcactaccaga tttgtttgacaggagcgacaat (SEQ ID NO: 7)(SEQ ID NO: 8) KLF4 CDS acccatccttcctgcccgatcagattggtaatggageggegggacttg (SEQ ID NO: 9) (SEQ ID NO: 10) KLF4 Placcacctcgccttacacatgaaga tagcgtaaaaggagcaacatag (SEQ ID NO: 11)(SEQ ID NO: 12) LMYC CDS gcgaacccaagacccaggcctgctcccagggggtctgctcgcaccgtgatg (SEQ ID NO: 13) (SEQ ID NO: 14) LMYC Plaggctgagaagaggatggctac tttgtttgacaggagcgacaat (SEQ ID NO: 15)(SEQ ID NO: 16) LIN28 CDS agccatatggtagcctcatgtccgctcaattctgtgcctccgggagcagggtagg (SEQ ID NO: 17) (SEQ ID NO: 18) LIN28 Plaagccatatggtagcctcatgtccgc tagcgtaaaaggagcaacatag (SEQ ID NO: 19)(SEQ ID NO: 20) RPL13A cctggaggagaagaggaaaga ttgaggacctctgtgtatttg(SEQ ID NO: 21) (SEQ ID NO: 22) B2M tgctgtctccatgtttgatgttctctgctccccacctctaag (SEQ ID NO: 23) (SEQ ID NO: 24) EBNA1atcagggccaagacatagaga gccaatgcaacttggacgtt (SEQ ID NO: 25)(SEQ ID NO: 26) EBNA2 catagaagaagaagaggatgaaga gtagggattcgagggaattactga(SEQ ID NO: 27) (SEQ ID NO: 28) LMP1 atggaacacgaccttgagatgagcaggatgaggtctagg (SEQ ID NO: 29) (SEQ ID NO: 30) BZLF1cacctcaacctggagacaat tgaagcaggcgtggtttcaa (SEQ ID NO: 31)(SEQ ID NO: 32) OriP tcgggggtgttagagacaac ttccacgagggtagtgaacc(SEQ ID NO: 33) (SEQ ID NO: 34) GAPDH accacagtccatgccatcactccaccaccctgttgctgta (SEQ ID NO: 35) (SEQ ID NO: 36) TDGFtccttctacggacggaactg agaaatgcctgaggaaagca (SEQ ID NO: 37)(SEQ ID NO: 38) NCAM1 gattcctcctccaccctcac caatattctgcctggcctggatg(SEQ ID NO: 39) (SEQ ID NO: 40) HAND1 ccacacccactcagagccattcaccccaccaccaaaacctt (SEQ ID NO: 41) (SEQ ID NO: 42) MSX1cgagaggaccccgtggatgcagag ggeggccatcttcagcttctccag (SEQ ID NO: 43)(SEQ ID NO: 44) AFP gaatgctgcaaactgaccacgctggaactggcattcaagagggttttcagtctgga (SEQ ID NO: 45) (SEQ ID NO: 46) SMN ctatcatgctggctgcct ctacaacacccttctcacag PCR-RFLP (SEQ ID NO: 47)(SEQ ID NO: 48)

Example 14 Cytogenetic Stability of PBMC vs. Fibroblast-Derived iPSCLines

TABLE 3A Cytogenetic Stability of PBMCs BC-iPSCs Description Numbers %abnormal Total iPSC lines 90 3.3% Total abnormal 3 *Total withnon-clonal aberration 3 3.3%

TABLE 3B Cytogenetic Stability of PBMCs Fibroblast-iPSCs DescriptionNumbers % abnormal Total iPSC lines 242 25.6% Total abnormal 62 *Totalwith non-clonal aberration 17 7.0%

Example 15 Cytogenetic Stability of PBMC vs. Fibroblast-Derived iPSCLines Array CGH

TABLE 4A Cytogenetic Stability of PBMCs BC-iPSCs Description NumbersTotal iPSC lines 7 De Novo* amp/dels 4 Average De Novo amp/dels 0.6 DeNovo amp/dels per passage 0.036

TABLE 4B Cytogenetic Stability of PBMCs Fibroblast-iPSCs DescriptionNumbers Total iPSC lines 6 De Novo amp/dels 19 Average De Novo amp/dels3.2 De Novo* amp/dels per passage 0.136

A variety of results demonstrating superior properties of BC-iPSCs areshown, including specific cell lines. High Success rate of peripheralblood mononuclear cell (PBMC)-based episomal reprogramming on MEFs isshown in Table 5. Frequency of different types of karyotypeabnormalities at individual; chromosomal level observed by G-bandkaryotyping in fibroblast-derived iPSCs. Rearr.—Rearrangement;Inv.—Inversion; Der.—derivatives is shown in Table 6. Success rate ofblood-based reprogramming on human recombinant laminin 521 in defined E7reprogramming media is shown in Table 7. Lack of success of blood-basedepisomal reprogramming on Matrigel substrate and defined E7reprogramming media is shown in Table 8. * De Novo or deletions arethose newly acquired amplifications or deletions during reprogrammingand/or expansion of the respective iPSC lines when compared to theparent donor tissue aCGH profile

TABLE 5 High Success rate of peripheral blood mononuclear cell(PBMC)-based episomal reprogramming on MEFs. Starting cell T - T -Success NT - NT - Success Line Condition Fresh/Frozen number PlasmidTotal Picked Effrciency rate Total Picked Efficiency rate 02iCTRMEFs/PESC Fresh 3.00E+06 4P 22 22 7.33E−04 1 3 3 1.00E−06 1 03iCTRMEFs/PESC Fresh 3.00E+06 4P 9 9 3.00E−04 1 8 8 2.67E−06 1 03iCTRMEFs/PESC Cryopreserved 5.00E+06 4P 5 5 1.00E−04 1 0 0 0.00E+00 014isALS- MEFs/PESC Cryopreserved 5.00E+06 5P 21 21 4.20E−04 1 2 24.00E−07 1 80iCTR- #2 MEFs/PESC Cryopreserved 5.00E+06 5P 10 10 2.00E−041 0 0 0.00E+00 0 51iALS- MEFs/PESC Cryopreserved 5.00E+06 5P 12 82.40E−04 1 2 2 4.00E−07 1 89iALS- MEFs/PESC Cryopreserved 5.00E+06 5P 1515 3.00E−04 1 14 14 2.80E−06 1 138iALS- MEFs/PESC Cryopreserved 5.00E+065P 5 5 1.00E−04 1 0 0 0.00E+00 0 152iALS MEFs/PESC Cryopreserved5.00E+06 5P 7 7 1.40E−04 1 0 0 0.00E+00 0 07iASD MEFs/PESC Cryopreserved5.00E+06 5P 18 18 3.60E−04 1 12 12 2.40E−06 1 53iALS- MEFs/PESCCryopreserved 5.00E+06 5P 9 8 1.80E−04 1 15 13 3.00E−06 1 SOD1A4V134iALS-C9 MEFs/PESC Cryopreserved 5.00E+06 5P 8 8 1.60E−04 1 12 122.40E−06 1 58iALS-C9 MEFs/PESC Cryopreserved 5.00E+06 5P 4 4 8.00E−05 12 2 4.00E−07 1 37iALS-C9 MEFs/PESC Cryopreserved 5.00E+06 5P 0 00.00E+00 0 NP NP 79iCTR MEFs/PESC Cryopreserved 5.00E+06 5P 0 0 0.00E+000 NP NP 98iALS- MEFs/PESC Cryopreserved 5.00E+06 5P 6 6 1.20E−04 1 3 36.00E−07 1 700iCTR- MEFs/PESC Cryopreserved 5.00E+06 5P 12 12 2.40E−04 19 9 1.80E−06 1 012iASD MEFs/PESC Cryopreserved 5.00E+06 5P 12 122.40E−04 1 1 1 2.00E−07 1 776iCLN6 MEFs/PESC Cryopreserved 5.00E+06 5P12 12 2.40E−04 1 5 5 1.00E−06 1 013iCTR MEFs/PESC Cryopreserved 5.00E+065P NP NP 12 12 2.40E−06 1 770iCLN6 MEFs/PESC Cryopreserved 5.00E+06 5P12 12 2.40E−04 1 12 12 2.40E−06 1 013iCTR MEFs/PESC Cryopreserved5.00E+06 5P NP NP 12 12 2.40E−06 1 XH7iCTR MEFs/PESC Cryopreserved5.00E+06 5P NP NP 12 12 2.40E−06 1 WP3iCTR MEFs/PESC Cryopreserved5.00E+06 5P NP NP 12 12 2.40E−06 1 FP5iCTR MEFs/PESC Cryopreserved5.00E+06 5P NP NP 1 1 2.00E−07 1 702iGAN MEFs/PESC Cryopreserved5.00E+06 5P NP NP 12 12 2.40E−06 1 116iFNDI- MEFs/PESC Cryopreserved5.00E+06 5P NP NP 12 12 2.40E−06 1 nxx 361iGAN- MEFs/PESC Cryopreserved5.00E+06 5P NP NP 6 6 1.20E−06 1 nxx 012iGAN- MEFs/PESC Cryopreserved5.00E+06 5P NP NP 6 6 1.20E−06 1 nxx 2EVPiALS- MEFs/PESC Cryopreserved5.00E+06 5P NP NP 6 6 1.20E−06 1 nxx 7YXLiALS- MEFs/PESC Cryopreserved5.00E+06 5P NP NP 4 4 8.00E−07 1 nxx 9XXZiALS- MEFs/PESC Cryopreserved5.00E+06 5P NP NP 2 2 4.00E−07 1 nxx 6MBUiALS- MEFs/PESC Cryopreserved5.00E+06 5P NP NP 6 6 1.20E−06 1 nxx 5XVDiALS- MEFs/PESC Cryopreserved5.00E+06 5P NP NP 6 6 1.20E−06 1 nxx 7MTJiALS- MEFs/PESC Cryopreserved5.00E+06 5P NP NP 6 6 1.20E−06 1 nxx 0JGZiALS- MEFs/PESC Cryopreserved5.00E+06 5P NP NP 6 5 1.20E−06 1 nxx 9ZZ5iALS- MEFs/PESC Cryopreserved5.00E+06 5P NP NP 6 6 1.20E−06 1 7AF6iALS- MEFs/PESC Cryopreserved5.00E+06 5P NP NP 9 7 1.80E−06 1 2UNGiALS- MEFs/PESC Cryopreserved5.00E+06 5P NP NP 6 6 1.20E−06 1 nxx 1DGFiALS- MEFs/PESC Cryopreserved5.00E+06 5P NP NP 7 7 1.40E−06 1 nxx 9GXDiALS- MEFs/PESC Cryopreserved5.00E+06 5P NP NP 6 6 1.20E−06 1 nxx 2FN3iALS- MEFs/PESC Cryopreserved5.00E+06 5P NP NP 6 6 1.20E−06 1 nxx 6PYDiALS- MEFs/PESC Cryopreserved5.00E+06 5P NP NP 6 6 1.20E−06 1 nxx Average 2.20E−04 90.00% 1.40E−0490.24% NP: Not performed T: T cell reprogramming method NT: non-T cellreprogramming method 4P/5P: reprogramming factors on 4 or 5 plasmids

TABLE 6 Frequency of different types of karyotype abnormalitie %Abnormalities Rank Chromosome Total Gain Loss Rearr. Inv. Der. TotalGain Loss Rearr. chr1 11.8% 15.8% 0.0% 13.9% 0.0% 0.0% 2 2 8 1 chr2 0.0%0.0% 0.0% 0.0% 0.0% 0.0% 21 13 8 16 chr3 4.9% 5.3% 7.7% 2.8% 0.0% 0.0% 96 3 11 chr4 2.1% 0.0% 0.0% 8.3% 0.0% 0.0% 12 13 8 4 chr5 0.0% 0.0% 0.0%0.0% 0.0% 0.0% 21 13 8 16 chr6 6.9% 10.5% 0.0% 5.6% 0.0% 0.0% 6 4 8 7chr7 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 21 13 8 16 chr8 4.2% 5.3% 0.0% 5.6%0.0% 0.0% 10 6 8 7 chr9 2.1% 0.0% 0.0% 8.3% 0.0% 0.0% 12 13 8 4 chr100.7% 0.0% 0.0% 2.8% 0.0% 0.0% 19 13 8 11 chr11 6.9% 13.2% 0.0% 0.0% 0.0%0.0% 6 3 8 16 chr12 11.1% 21.1% 0.0% 0.0% 0.0% 0.0% 3 1 8 16 chr13 13.2%7.9% 7.7% 13.9% 100.0% 100.0% 1 5 3 1 chr14 5.6% 5.3% 0.0% 11.1% 0.0%0.0% 8 6 8 3 chr15 3.5% 2.6% 7.7% 2.8% 0.0% 0.0% 11 11 3 11 chr16 1.4%2.6% 0.0% 0.0% 0.0% 0.0% 15 11 8 16 chr17 1.4% 0.0% 7.7% 0.0% 0.0% 0.0%15 13 3 16 chr18 1.4% 0.0% 7.7% 0.0% 0.0% 0.0% 15 13 3 16 chr19 1.4%0.0% 0.0% 5.6% 0.0% 0.0% 15 13 8 7 chr20 2.1% 0.0% 0.0% 8.3% 0.0% 0.0%12 13 8 4 chr21 8.3% 5.3% 23.1% 5.6% 0.0% 0.0% 5 6 2 7 chr22 0.7% 0.0%0.0% 2.8% 0.0% 0.0% 19 13 8 11 chrX 10.4% 5.3% 38.5% 2.8% 0.0% 0.0% 4 61 11

TABLE 7 Success rate of blood-based reprogramming on human recombinantlaminin 521 02iCTR MEFs/PESC Fresh 3.00E+06 4P 22 22 7.33E−04 1 3 31.00E−06 1 02iCTR L521/E7 Fresh 3.00E+06 4P 3 3 1.00E−04 1 1 1 3.33E−051 03iCTR L521/E7 Cryopreserved 5.00E+06 5P 19 12 3.80E−04 1 34 126.80E−04 1 14isALS- L521/E7 Cryopreserved 5.00E+06 4P 0 0 0.00E+00 0 0 00.00E+00 0 80iCTR- L521/E7 Cryopreserved 5.00E+06 5P 4 4 8.00E−05 1 0 00.00E+00 0 80iCTR-#2 L521/E7 Cryopreserved 5.00E+06 5P NP NP NP 0 00.00E+00 0 89iALS- L521/E7 Cryopreserved 5.00E+06 5P 49 15 9.80E−04 1 00 0.00E+00 0 138iALS- L521/E7 Cryopreserved 5.00E+06 4P 0 0 0.00E+00 0 00 0.00E+00 0 07iCTR- L521/E7 Cryopreserved 5.00E+06 5P 49 16 9.80E−04 1102 18 2.04E−03 1 179iCTR- L521/E7 Cryopreserved 5.00E+06 5P NP NP NP 11 2.00E−05 1 201iCTR- L521/E7 Cryopreserved 5.00E+06 5P 6 6 1.20E−04 1 44 8.00E−05 1 202iCTR- L521/E7 Cryopreserved 5.00E+06 5P 0 0 0.00E+00 0 44 8.00E−05 1 206iCTR L521/E7 Cryopreserved 5.00E+06 5P 5 5 1.00E−04 1 NPNP NP 166isALS L521/E7 Cryopreserved 5.00E+06 5P 5 5 1.00E−04 1 NP NP NP276iCTR L521/E7 Cryopreserved 5.00E+06 5P 12 12 2.40E−04 1 36 127.20E−04 1 6PYDiALS- L521/E7 Cryopreserved 5.00E+06 5P NP NP NP 6 61.20E−04 1 nxx Average 2.57E−04 75.00% 2.90E−04 61.54% NP: Not performedT: T cell reprogramming method NT: non-T cell reprogramming method4P/5P: reprogramming factors on 4 or 5 plasmids

TABLE 8 Relative lack of success of blood-based episomal reprogrammingon Matrigel substrate CS021CTR MG/LCLRM Fresh 3.00E+06 4p 0 0 0.00E+00 00 0 0.00E+00 0 CS031CTR MG/E7 Frozen 5.00E+06 4p 0 0 0.00E+00 0 0 00.00E+00 0 6PYDiALS 2[Mp]/E7 Frozen 5.00E+06 5p NP NP 0 0 0.00E+00 0Average 2.57E−04 75.00% 2.90E−04 61.54% NP: Not performed T: T cellreprogramming method NT: non-T cell reprogramming method 4P/5P:reprogramming factors on 4 or 5 plasmids

Example 16 PBMCs Isolated from a Blood Draw are Reliably Reprogrammed toiPSCs

Dermal fibroblasts obtained from a skin biopsy have been the most commonsource of iPSC reprogramming material, due largely to their inexpensiveand relatively easy usage. However, compared to a blood draw, a skinpunch biopsy can be between the size of 3-4 mm and is often a painfulprocedure that requires local anesthetics. Compared to skinbiopsy-derived fibroblasts, blood is a more accessible source of patienttissue during hospital visits and, therefore, a preferred choice ofsource tissue for iPSC reprogramming by both patients and clinicians.Various human blood reprogramming methods have been reported with mixedefficiencies, including CD34⁺ cells mobilized from peripheral blood(ref). Unless isolated from cord blood, CD34⁺ cells are usually isolatedfrom the peripheral blood or bone marrow of patients undergoing G-CSFmobilization for several days. In order to generate sufficient cellnumbers for reprogramming, isolated CD34⁺ progenitor cells need to beenriched and expanded in culture with complex and expensive protocols.The Inventors were successful in reprogramming PBMCs that were freshlyisolated from a peripheral blood draw without prior cell expansion orCD34⁺ cell isolation using episomal reprogramming plasmids. Such anapproach provides the fastest and most cost effective procedure forobtaining a patient's reprogrammed iPSCs from the time of collecting apatient bio-specimen to generating their rigorously characterized iPSCs.

Reprogramming large cohorts of PBMCs from multiple subjectssimultaneously as well as iPSC line generation at a much later dateafter a patient blood draw that allows for iPSC “future-proofing”,requires a PBMC cryopreservation step to recover viable cells reliably.Additionally, this process is flexible because selective cohorts of wellcryopreserved PBMCs from representative patients could be converted toiPSCs when greater patient genotype-phenotype information is available.However, when cryopreserved PBMCs from multiple individuals werereprogrammed to iPSCs with episomal plasmids expressing POU5F1, SOX2,KLF4, LIN28, L-MYC, p53 shRNA, the Inventors observed significantvariability in isolating identifiable iPSC clones even after 35-40 days,regardless of PBMC cell type, ECM substrata and media). The Treprogramming method was successful with only 33% of PBMC samples, whilethe non-T method on cryopreserved PBMCs did not result in any clonaliPSC lines (data not shown).

Critically, the reliability of PBMC reprogramming in our hands wasimproved significantly when the Inventors utilized, (a) two additionalepisomal plasmids containing SV40 large T antigen (SV40LT) and EBNA-1 inspecific stoichiometry to minimize PBMC cell death and sustainreprogramming factor plasmid expression and (b) a defined reprogrammingmedia to promote high surface attachment of the nucleofected PBMCs (FIG.8A). This novel protocol resulted in successful and efficient generationof multiple adherent PBMC-iPSC clones that could be mechanicallyisolated and scaled up for expansion after 25-35 days post-nucleofection(FIG. 8A). Importantly, the success rate in reprogramming multipleindividual cryopreserved PBMCs from unaffected controls or diseasedpatients' was 90% for the T cell method and 83% for the non-T cellmethod (Table 5) on mouse embryonic fibroblast feeders (MEFs). Given thelimitations imposed by T cell-iPSCs and the low efficiencies forPBMC-derived non-T cell reprogramming, this method resulted in reliablereprogramming of non-T cell derived BC-iPSCs.

The Inventors next tested whether this new protocol was also amenablewhen using reprogramming methods that are more clinically compatible.Use of recombinant human laminin 521 substrate and chemically definedreprogramming media resulted in successful PBMC-reprogramming, albeit ata slightly lower success rate at 75% and 58%, respectively. The averagereprogramming efficiency for T-cell and non-T cell method was 2.2×10⁻⁴%and 1.6×10⁶%, respectively (Table 5). All the PBMC-iPSC lines exhibitedtypical PSC characteristics, including tightly packed colonies, highcell nuclear-cytoplasmic ratio, robust alkaline phosphatase activity,and expression of pluripotency antigens (FIG. 8B). The BC-iPSCs passedpluripotency quality control metrics determined by the PluriTest assay,demonstrating that the PBMC-iPSC transcription profile was analogous towell established hESCs and fib-iPSCs, but not differentiated fibroblastsand neural progenitor cells (FIG. 8C). BC-iPSCs maintained a normalG-band karyotype (FIG. 8D) and were confirmed to be clonal derivativesof either T cells or non-T cells from the PBMCs using the T cellreceptor (TCR)-β and -γ, gene rearrangement/clonality assays (FIG. 8E).The trilineage potential of BC-iPSCs was demonstrated by spontaneousembryoid body formation and by measuring germ-layer specific geneexpression profile by the TaqMan Scorecard assay (FIG. 8F). The“footprint-free” status of BC-iPSCs was confirmed by demonstration ofendogenous pluripotency gene expression and absence of exogenousreprogramming transgenes using RT-qPCR (FIG. 11A). The EBNAplasmid-related latency element was eventually eliminated from theestablished BC-iPSCs (FIG. 11B).

Example 17 BC-iPSCs have Equivalent Neuronal Differentiation Compared toFib-iPSCs

It has been reported that iPSCs, regardless of the source tissue,ultimately lose most of their gene expression and epigenetic profilesrelated to the original cell source. However, it remains unclear whetherblood-derived iPSCs can differentiate as efficiently asfibroblast-derived iPSCs into other various cell types, possibly due toa stronger retention of epigenetic memory in blood-sourced iPSCs. TheInventors addressed this by directing blood or fibroblast-derived iPSCs,both of mesodermal origin, to a different germ layer such as ectoderm.Specifically, the Inventors performed neural ectoderm differentiationfrom large numbers of fibroblast (n=26) and PBMC lines (n=8), from bothhealthy volunteers as well as diseased patients. Based onimmunocytochemistry for neural ectoderm markers of TUBB3 (β₃-tubulin)and NKX6.1 and subsequent cell counts, neuronal differentiation wasshown to occur at a similar efficiency between fibroblast and BC-iPSCs(FIG. 8G), further demonstrating the utility of BC-iPSCs.

Example 18 BC-iPSCs Maintain a Significantly More Stable KaryotypeCompared to Fib-iPSCs

Recurrent chromosomal abnormalities have been described previously foran abundant number of hESC lines. A few reports have chronicled commonchromosomal aberrations for hiPSC lines, however, these did notmethodically account for variability in source tissues, reprogrammingmethods and cell culture methods. To our knowledge, there have been nosystematic studies describing cytogenetic analysis comparing frequencybetween iPSCs derived from fibroblast and blood. Over the past six yearsthe Inventors performed routine cytogenetic analysis on iPSC linesderived from 104 unique fibroblast or PBMC donors, which includes 339independent clonal iPSC lines. All fibroblast and blood-derived iPSCsassessed were reprogrammed using the similar non-integrating episomalreprogramming and standard feeder-free matrigel/mTeSR cell culturemethods.

Although fib-iPSCs and BC-iPSCs derived in the iPSC Core resemble eachother with regard to their reprogramming methods, pluripotency anddifferentiation, an early observation of striking differences incytogenetic stability between the two source cell types prompted theneed for large-scale comparative analyses. Many laboratories and stemcell repositories perform routine quality control assays of cytogeneticchanges in PSCs using G-band karyotyping by obtaining cell metaphasesand Giemsa staining of the chromosomes. This method can readily identifysmall subpopulations of abnormal cells where reliable identification of5% abnormal cells is readily achieved, thus allowing detection ofmosaicism (>10%) and balanced translocations at low resolution (>5-10Mb).

The Inventors performed karyotype analysis on 364 human iPSC cultureswhere source fibroblasts and PBMCs were collected from multiplelaboratories or public repositories. Our data reveal remarkabledifferences in the incidence of chromosomal aberrations in fib-iPSCs andBC-iPSCs. Abnormal karyotypes with clonal aberrations were observed in59 of 258 (22.9%) cultures from clonally independent human fib-iPSClines, derived from 78 unique donors (FIG. 6). In stark contrast, only 4in 106 (3.8%) cultures of clonal human PBMC-iPSC lines derived from 32unique donors displayed low-frequency abnormalities. It is evident fromthe ideograms, schematic representation of the chromosomes, thatrecurrent aberrations are represented in much greater degree in infib-iPSCs compared to the BC-iPSCs (FIG. 6). Indeed, this remarkablecytogenetic stability is unique to PBMCs, as iPSCs reprogrammed fromother cell origins, including lymphoblastoid cell lines (25%) andprimary epithelial cells (27%) have similar high rates of karyotypesaberrations (FIG. 12).

The most frequent karyotypic abnormalities in fib-iPSCs were observed inchromosomes 13 (13.2%), 1 (11.8%), 12 (11.1%), and X (10.4%) (Table 6).To a lesser extent karyotype changes were also observed in chromosomes21, 11, and 6, in descending order of frequency. Of the aneuploidies,chromosomal gains (trisomy or duplications) were most commonly observedin 12 (21.1%), 1 (15.8%), 11 (13.2%), and 6 (10.5%) and chromosomallosses were repeatedly observed in chromosomes X (38.5%) and 21 (23.1%).Translocation rearrangements in chromosomes 1 and 13 (13.9%) and 14(11.1%) in fib-iPSCs were also observed. BC-iPSCs had one line eachdisplay chromosome gains in 14 (trisomy) or 18 (duplication). The othertwo PBMC-iPSC lines had exhibited uncommon abnormalities: atranslocation 46,XY,t(7;14)(q34;q11.2) and a mosaic deletion46,X,del(X)(q22q26) in 19% of cells (Table 6).

Example 19 Fib- and BC-iPSCs Karyotype Distribution Relates not to theDonor Age but Rather to Passage Number

The skin, unlike the blood, is routinely exposed to externalenvironmental elements like potential sun damage and the fibroblastsderived from the biopsies require a certain level of expansion prior toreprogramming, which is akin to further aging in culture. Given this,the Inventors posited that the donor age might be a contributing factorthat impacts the cytogenetic instability of fib-iPSCs. However, withrespect to the frequency of cytogenetic abnormalities in fib-iPSCs, theInventors did not observe a significant trend or correlation with theage of the donor (FIG. 9A). Indeed, based on G band karyotype analysis,the highest percent of karyotype abnormalities (31.9%) was observed inthe 21-40 age group fibroblast-iPSCs.

Pluripotent stem cells have a highly proliferative nature. As such, theInventors next attempted to analyze how the fib- and BC-iPSCs fared overtime in cell culture and whether increasing passage number led to agreater propensity for accumulating cytogenetic aberrations. About 21.6%of fibroblast-derived iPSCs were observed to have abnormal karyotypes(>1/20 cells with clonal aberrations) in their first G-band karyotypeevaluation post-iPSC generation, which was typically between passages4-23. In stark contrast, only 2.8% of BC-iPSCs had abnormal karyotypesin their first assessment. Any iPSC line that was determined to have anabnormal karyotype was no longer cultured or evaluated. Therefore,between 2^(nd) and 4^(th) repeat karyotypes were only evaluated on iPSClines that were initially identified as cytogenetically normal in theirfirst karyotype. This analysis allowed us to determine whether fib-iPSCshave an inherent level of cytogenetic instability over BC-iPSCs uponcontinued time in culture and passaging even after being established asnormal post-iPSC generation (at their first G-band karyotypeevaluation). G-band karyotyping showed that a greater proportion offib-iPSCs (27.8%) acquired abnormal karyotypes in culture upon repeatkaryotyping when compared to the proportion at first karyotypes(P<0.001, two way ANOVA with Bonferroni posttest), while the percentabnormal karyotypes in BC-iPSCs did not show any significant differencebetween first and repeat karyotypes (FIG. 9B). Upon analyzing the originof the cytogenetic instability in fib-iPSCs, it appeared that thehighest rate of abnormal karyotypes (first or repeat) occur duringpassages 10-23 in the life of the iPSC line (FIG. 9C-D), which istypically when the largest amount of iPSC expansion occurs for any givencell line. The iPSC expansion increases at passages 10-23 largely due tocharacterization and cell banking coinciding around these passagenumbers. Nevertheless, this is a similar process for both fib- orBC-iPSCs, and yet the PBMC-derived cells do not display an increaseddisposition towards abnormal karyotypes upon extended culture orexpansion.

Example 20 BC-iPSCs Acquire Less Submicroscopic Amplifications andDeletions

G-band karyotype has the advantage that it can detect balancedtranslocations, inversions, <20% culture mosaicism, chromosomal positionof genomic gains or losses as commonly observed in the fib-iPSC cultures(FIG. 6 and Table 5). However, since G-band karyotype is unable todetect submicroscopic genomic abnormalities (<5 Mb), the Inventorsdecided to conduct comparative genomic hybridization (aCGH) microarrayon a subset of fib-iPSC and PBMC-iPSC lines. While aCGH is unable todetect balanced translocations, inversions and <20% culture mosaicism,it is a good supportive method to detect smaller size genomic gains andlosses, copy number variants (CNVs), duplications/deletions, andunbalanced translocations in the iPSC lines. The Inventors analyzed andrecorded any de novo amplifications or deletions acquired in the iPSCsupon comparison with the parental fibroblast or PBMC sourcebio-specimen. In this analysis only those amplifications and deletionsacquired de novo in iPSCs were considered, when they were not normalCNVs upon cross referencing against the Database of Genomic Variants(DGV) that contains genomic variations observed in healthy individuals.

Including iPSC lines with abnormal and normal G-band karyotypes, theaverage size of the amplification and deletions detected by aCGH weresignificantly greater in fib-iPSC lines at 44 Mb compared to 2.1 Mb inPBMC-iPSC lines (FIG. 10A), the preponderance of which was due tofib-iPSC lines with an abnormal karyotype (FIG. 10B). Upon segregatingthe size analysis comparison between iPSC lines with normal karyotypes,the de novo amps/dels were on average 2.31 Mb in fib-iPSCs and 2 Mb inPBMC-iPSC lines. Supporting this data, the average number of acquired denovo total amps/dels in fib-iPSCs (3.7) were at least two-fold greaterthan in BC-iPSCs (1.8). Even in iPSC lines that were determined to havenormal G-band karyotypes, the number of new amps/dels were greater infib-iPSCs at 3.3 vs. BC-iPSCs at 1.8. The most commonly acquiredsubmicroscopic (0.8-1.5 Mb) de novo amplifications or deletions in fib-or PBMC-iPSC lines detected by aCGH was amplification of chromosome7q31.32 or deletion of chromosomes 10q15.2-q25.1, 16p11.2, 21p11.2-p11.1(FIG. 10E).

Here the Inventors report a new and more reliable method forreprogramming of the non-T cell component of blood using episomalplasmids expressing pluripotency factors. Using the describedreprogramming protocol, one is able to consistently reprogram non-Tcells with close to 100% success from non-T cell or non-B cell sources.Further advantages include use of a defined reprogramming media E7 andusing defined clinically compatible substrate recombinant human L-521.Blood cell-derived iPSCs (“BC-iPSCs”) exhibited identicalcharacteristics to fibroblast derived iPSCs (“fib-iPSCs”), retaingenotype, exhibit a normal pluripotency profile, and readilydifferentiate into all three germ-layer cell types. This method forreliably deriving iPSCs from patient blood samples paves the way forrapidly generate new human iPSC lines, including patient-specific lines,thus providing an enormous bioresource for disease modeling, drugdiscovery, and regenerative medicine applications.

The various methods and techniques described above provide a number ofways to carry out the invention. Of course, it is to be understood thatnot necessarily all objectives or advantages described may be achievedin accordance with any particular embodiment described herein. Thus, forexample, those skilled in the art will recognize that the methods can beperformed in a manner that achieves or optimizes one advantage or groupof advantages as taught herein without necessarily achieving otherobjectives or advantages as may be taught or suggested herein. A varietyof advantageous and disadvantageous alternatives are mentioned herein.It is to be understood that some preferred embodiments specificallyinclude one, another, or several advantageous features, while othersspecifically exclude one, another, or several disadvantageous features,while still others specifically mitigate a present disadvantageousfeature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be mixed andmatched by one of ordinary skill in this art to perform methods inaccordance with principles described herein. Among the various elements,features, and steps some will be specifically included and othersspecifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the invention extend beyond the specificallydisclosed embodiments to other alternative embodiments and/or uses andmodifications and equivalents thereof.

Many variations and alternative elements have been disclosed inembodiments of the present invention. Still further variations andalternate elements will be apparent to one of skill in the art. Amongthese variations, without limitation, are sources of blood cells,cellular components of blood, pluripotent stem cells derived fromtherein, techniques and composition related to deriving pluripotent stemcells from blood cells cells, differentiating techniques andcompositions, biomarkers associated with such cells, and the particularuse of the products created through the teachings of the invention.Various embodiments of the invention can specifically include or excludeany of these variations or elements.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment ofthe invention (especially in the context of certain of the followingclaims) can be construed to cover both the singular and the plural. Therecitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations on those preferred embodiments will become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Itis contemplated that skilled artisans can employ such variations asappropriate, and the invention can be practiced otherwise thanspecifically described herein. Accordingly, many embodiments of thisinvention include all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that can be employed can be within thescope of the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention can be utilized inaccordance with the teachings herein. Accordingly, embodiments of thepresent invention are not limited to that precisely as shown anddescribed.

The invention claimed is:
 1. A method of generating blood cell derivedinduced pluripotent stem cells, comprising: providing a quantity ofblood cells; delivering a quantity of EBNA1 and reprogramming factorscomprising Oct-4, Sox-2, Klf-4, 1-Myc, Lin-28, SV40 Large T Antigen(“SV40LT”), and short hairpin RNAs targeting p53 (“shRNA-p53”) into theblood cells; and culturing the blood cells in a reprogramming media forat least 4 days, wherein delivering the EBNA1 and reprogramming factors,and culturing in a reprogramming media generates blood cell derivedinduced pluripotent stem cells, wherein the reprogramming factors areencoded in four oriP/EBNA1 derived vectors comprising: a first vectorencoding Oct4, Sox2, SV40LT and Klf4, a second vector encoding Oct4 andshRNA-p53, a third vector encoding Sox2 and Klf4, and a fourth vectorencoding 1-Myc and Lin-28; and wherein a fifth oriP/EBNA1 derived vectorencodes EBNA1.
 2. The method of claim 1, wherein delivering a quantityof reprogramming factors comprises nucleofection.
 3. The method of claim1, wherein the five oriP/EBNA1 derived vectors are pEP4 E02S ET2K,pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCWB-EBNA1.
 4. Themethod of claim 1, comprising plating of the blood cells on a treatedcell culture surface after delivering reprogramming factors into theblood cells, and culturing the blood cells in a reprogramming media onsaid treated cell culture surface.
 5. The method of claim 4, wherein thetreated cell culture surface comprises plating of mouse embryonicfeeders (MEFs).
 6. The method of claim 4, wherein the treated cellculture surface comprises an extracellular matrix protein.
 7. The methodof claim 6, wherein the extracellular matrix protein comprises laminin.8. The method of claim 7, wherein laminin comprises L-521.
 9. The methodof claim 1, wherein the reprogramming media comprises embryonic stemcell (ESC) media.
 10. The method of claim 9, wherein the ESC mediacomprises basic fibroblast growth factor (bFGF).
 11. The method of claim1, wherein the reprogramming media comprises E7 media.
 12. The method ofclaim 11, wherein the reprogramming media comprises E7 media comprisingL-Ascorbic Acid, Transferrin, Sodium Bicarbonate, Insulin, SodiumSelenite and/or bFGF.
 13. The method of claim 1, wherein culturing theblood cells in a reprogramming media is for at least 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, or 16 days.
 14. The method of claim 1, whereinculturing the blood cells in a reprogramming media is for at least 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days.
 15. Themethod of claim 1, wherein the blood cells are isolated from a subjectpossessing a disease mutation.
 16. The method of claim 15, wherein thedisease mutation is associated with a neurodegenerative disease,disorder and/or condition.
 17. The method of claim 15, wherein thedisease mutation is associated with an inflammatory bowel disease,disorder, and/or condition.
 18. The method of claim 1, wherein bloodcells are non T-cell, non B-cell mononuclear cells.
 19. The method ofclaim 1, wherein the blood cells are a sample drawn from a humansubject.
 20. The method of claim 19, wherein the sample is whole blood.21. The method of claim 19, wherein the sample is peripheral blood. 22.The method of claim 19, wherein the sample is an isolated component ofnon T-cell, non B-cell mononuclear cells.
 23. The method of claim 1,wherein the blood cells are from a previously frozen sample thawed priorto providing a quantity of blood cells.
 24. The method of claim 1,wherein the blood cells are not expanded prior to delivering thequantity of EBNA1 and reprogramming factors.